Chemistry and Biology of Pteridines: 8 Montreal, Canada, June 15–20, 1986 9783110856262, 9783110107715

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Chemistry and Biology of Pteridines 1986 Pteridines and Folic Acid Derivatives

Chemistry and Biology ofPteridines 1986 Pteridines and Folic Acid Derivatives Proceedings of the Eighth International Symposium on Pteridines and Folic Acid Derivatives Chemical, Biological and Clinical Aspects Montreal, Canada, June 15-20,1986 Editors B. A. Cooper • V M.Whitehead

w G DE

Walter de Gruyter • Berlin • New York 1986

Editors

Professor V Michael Whitehead Hematology Service Montreal Children's Hospital 2300 Tupper Street Montreal, Québec H3H 183 Canada

Professor Bernard A. Cooper Division of Hematology and Medical Oncology Royal Victoria Hospital 687, Avenue des Pins Ouest Montréal, Québec H3A 1A1 Canada

Library of Congress Cataloging in Publication Data International Symposium on Pteridines and Folic Acid Derivatives: Chemical, Biological, and Clinical Aspects (8th : 1986 : Montréal, Québec) Chemistry and biology of pteridines, 1986. Includes bibliographies and indexes. 1. Pteridines-Congresses. 2. Folic acid-Derivatives-Congresses. I. Cooper, B. A. (Bernard A.), 1928- . II. Whitehead, V M. (V Michael), 1934III. Title. [DNLM: 1. Folic Acid-analogs & derivativescongresses. 2. Pteridines-congresses. W3 IN922J 8th 1986c / QU 188 1616 1986c] QP801.P69I585 1986 612'.399 86-19888 ISBN 0-89925-271-0

CIP-Kurztitelaufnahme

der Deutschen

Bibliothek

Chemistry and biology of pteridines . . . : pteridines and folic acid derivatives ; proceedings of the . . . Internai. Symposium on Pteridines and Folic Acid Derivatives, Chem., Biolog. and Clin. Aspects. - Berlin ; New York : de Gruyter Bd. 5, Kongressname: Internat. Symposium on Chemistry and Biology of Pteridines NE: International Symposium on Pteridines and Folic Acid Derivatives, Chemical, Biological and Clinical Aspects; International Symposium on Chemistry and Biology of Pteridines 8.1986. Montreal, Canada, June 15-20. 1986. -1986. ISBN 3-11-010771-6

Copyright © 1986 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Gerike G m b H , Berlin. - Binding: D. Mikolai, Berlin. - Printed in Germany.

PREFACE These proceedings are based on the scientific presentations made at the 8th International Symposium: Pteridines and Folic Acid Derivatives, held in Montreal, Canada, June 15-20, 1986.

In

designing this symposium, the organizers perpetuated many features of earlier meetings, including plenary session format, state-of-the-art lectures, the F. Gowland Hopkins Lecture and emphasis on poster presentations.

An innovation of the program

was the introduction of plenary poster discussion sessions in which a knowledgeable scientist stimulated discussion on a collection of posters after they had been on view for some time.

These proved lively and instructive and were

well-attended.

Chairmen of sessions were invited to provide

brief summaries of the areas addressed.

These form part of

these proceedings.

A welcoming reception was held at the McCord Museum and a symposium banquet at Heléne de Champlain restaurant.

The latter

-

coincided with the all-nations finale of the Montreal Festival of Fireworks, which added some excitement to the evening.

We thank Prof. Hamish Woods, organiser of the 7th International Symposium, for advice, details of planning and process, including a mailing list and for a generous donation to start our activities.

We thank other generous donors listed under

acknowledgments and members of the Advisory Committee, the Folate Club of Montreal, the Social Committee and the staff at the McGill Conference Centre, in particular Charlotte

VI

Benabdullah, Jean Lenzi and Joan Gross, for contributing to the success of the symposium.

We thank Prof. Adrien Albert for

preparing a brief history of the pteridine symposia included in these proceedings. Finally, we thank the participants in the symposium whose productive research and enthusiasm to exchange knowledge made this meeting the success it was.

Bernard A. Cooper Montreal, June 1986

V. Michael Whitehead

ACKNOWLEDGMENTS

The Committee gratefully acknowledges contributions from the following sponsors: Air Canada Bristol-Meyers Company, New York, U.S.A. Burroughs Wellcome Co., North Carolina, U.S.A. 7th International Pteridine Symposium, The University of Strathclyde, Scotland Lederle Laboratories, New York, U.S.A. Lederle Laboratories, Ontario, Canada McGill University - Montreal Children's Hospital Research Institute Medical Research Council of Canada, Government of Canada Moravek Biochemicals, Inc., California, U.S.A. National Health and Welfare, Government of Canada National Cancer Institute, National Institute of Health, Maryland, U.S.A. Medical Liaison Service, Sandoz Canada Inc., Quebec, Canada Warner Lambert Co., Michigan, U.S.A.

CONTENTS

How it all Began: A Brief History of the Pteridine Symposia A. Albert

1

The Gowland Hopkins Lecture: The Mechanisms of Action of Folate and Pterin Requiring Enzymes S.J. Benkovic, L.J. Slieker, S.C. Daubner, L.F. Courtney, T.A. Dix, S.O. Pember, L.M. Bloom, C.A. Fierke, R.J. Mayer, J.-T. Chen, K. Taira

13

SECTION A: CHEMISTRY OF PTERINS AND FOLATES

Side-Chain Chemistry of Pteridines W. Pfleiderer, V. Kang, R. Soyka, W. Hutzenlaub, M. Wiesenfeldt, W. Leskopf

31

A General Method for the Synthesis of 10-Substituted-10-Deaza Folate Analogues M.G. Nair, T.R. Toghiyani, B. Ramamurthy, R.L. Kisliuk, Y. Gaumont

45

Synthesis and Properties 'of Pyrimidodiazepines as Ring-Strained Probes of Pterin Utilizing Enzymes S.W. Bailey, D.C. Pike, J.E. Ayling

51

Summary: Pterin and Folate Chemistry E. Taylor A Facile Method for Preparing

55 2,4-Diamino-5,8-Dideazapterins

J.B. Hynes, A. Pathak, C.H. Panos, C.C. Okeke

57

Synthesis of 5,10-Dideaza-5,6,7,8-Tetrahydrofolic Acid (DDATHF) and Analogs E.C. Taylor, G.S.K. Wong, S.R. Fletcher, P.J. Harrington, G.P. Beardsley, C.J. Shih

61

Synthetic Routes to Methotrexate- and Folic Acid-Polyglutamates of High Chemical and Enantiomeric Purity M. Przybylski, R. Renkel, P. Fonrobert

65

X In Vivo and In Vitro Evaluations of 5-[4-(Substituted Aryl)-1Piperazinyl]-6-Alkyl-2,4-Pyrimidinediamines as Antitumor Agents L.M. Werbel, J. Hung, J.A. Besserer, T.J. Boritzki, W.R. Leopold, D.W. Fry

The Electrochemical

Oxidation of Tetrahydropterin

Derivatives

S. Kwee

A Practical

69

73

Synthesis of (6R)- and

(6^)-5,6,7,8-Tetrahydrobiopterins

S. Matsuura, T. Sugimoto, S. Murata

77

Chemical Synthesis and Properties of Quinonoid

(6Rj-Dihydrobiopterin

S. Matsuura, S. Murata, T. Sugimoto

81

Pyrazine-Ring Conformations of Tetrahydropterins and QuinonoidDihydropterins J.E. Gready

85

Hydrogénation of Silylated Pterins in Benzene Solution P.H. Boyle, M.F. Kelly

N(5)-A1kylation of Polyacetylated

91

5,6,7,8-Tetrahydroneopterins

S. Antoulas, M. Viscontini

Oxidations Photosensitized by Pterins and

95

Diaminopteridines

M. Aubailly, R. Santus

99

Structure of the Riboflavin Synthase/Lumazine Synthase Complex. Arrangement and Chain Folding of Subunits in the Icosahedral Capsid R. Ladenstein, H.D. Bartunik, M. Schneider, R. Huber, K. Schott, A. Bacher

Use of C18 Silica Cartridges to Purify and Characterize

103

Pterins

K.B. Jacobson, J. Ferre

107

Synthesis and Structure-Activity Relationships of Tetrahydrobiopterin Cofactor Analogues E.C. Bigham, G.K. Smith, J.F. Reinhard, Jr

111

XI Synthesis, Purification and Properties of a New Class of Pteridine Derivatives J. Ferre, J.J. Yim, W. Pfleiderer, K.B. Jacobson

115

Summary: Chemistry of Pterins J.B. Hynes

121

S E C T I O N B: B I O S Y N T H E S I S A N D B I O C H E M I S T R Y OF

PTERINS

Biosynthesis of Tetrahydrobiopterin and Related Compounds in Drosophila melanogaster G.M. Brown, J.P. Primus, A.C. Switchenko

125

Tetrahydrobiopterin Biosynthesis in Man H.-C. Curtius, S. Takikawa, A. Niederwieser

The Biosynthesis of Tetrahydrobiopterin

in the Bovine Adrenal

141

Medulla

G.K. Smith, D.S. Duch, C.A. Nichol

The Biosynthesis of Tetrahydrobiopterin

151

in Rat Brain

S. Milstien, S. Kaufman

169

Summary: Biosynthesis of Tetrahydrobiopterin G.M. Brown

183

The Metabolic Role of Tetrahydrobiopterin S. Kaufman

185

Spectroscopic Studies on Tyrosine 3-Monooxygenase from Bovine Adrenal Medulla: A Blue Iron Protein J. Haavik, K.K. Andersson, L. Petersson, T. Flatmark

201

Queuine-Lacking tNRAs in Relation to the Grade of Malignancy of Specific Tumors, Possible Role of Pteridines W. Kersten, E. Zubrod, B. Emmerich, P. Maubach

205

XII Pteridine Synthesis During Interaction of Interleukin 2 with T Lymphocytes - Modulator Function in IL-2 Signal Transmission I. Ziegler, J. Ellwart, U. Schwulera

209

SECTION C: BIOLOGY OF PTERINS

Dopamine Neurons in Culture: A Model System for Examination of Neuronal Biopterin Metabolism G. Kapatos, L.A. Chiodo

215

Role of the Pituitary in the Regulation of Tetrahydrobiopterin synthesis in the Non-Neuronal Tissues

Bio-

D.S. Duch, S.W. Bowers, C.A. Nichol

219

Distribution of GTP Cyclohydrolase-1, Neopterin, and Biopterin in the Human Brain T. Nagatsu, M. Sawada, M. Akino, M. Masada, T. Sugimoto, S. Matsuura

Enzymatic Synthesis of

223

6,7-Dimethyl-8-Ribityllumazine

A. Bacher, G. Neuberger, R. Volk

227

A Model for Hyperphenylal aninaemia Due to Tetrahydrobiopterin

Deficiency

R.G.H. Cotton

231

The Effect of Biopterin and its Reduced Derivatives on Cultured Human Lymphocytes S. Webber, D.L. Jaye

235

Developmental Aspects of Biopterin Metabolism in Human J.L. Dhondt, J.M. Hayte, G. Forzy, M. Delcroix, J.P. Farriaux, C. Largil 1 iere

Screening for Biopterin Defects Among Hyperphenylalaninemic Report of a Canadian Program After 3 Years

239

Patients:

F. Mohyuddin, C.A. Rupar, M.C. Evers

Need for Therapy i_n utero of Fetuses with Tetrahydrobiopterin

243

Deficiency

Y. Sawada, H. Shintaku, I. Suyama, G. Isshiki, Y. Hase, Y. Okano, H. Yamamoto, T. Tsuruhara, T. Oura

247

XIII P t e r i d i n e Metabolism in P a t i e n t s S u f f e r i n g from Amyotrophic Lateral S c l e r o s i s H.-J. Z e i t l e r ,

B. Andondonskaja-Renz, G. Küther, A. S t r u p p l e r

U r i n a r y Neopterin in Cocaine-Abusing

251

Individuals

R.W. Guynn, K. B i e h l , D.K. M e r r i l l , K.J. Krajewski Features of Neopterin Determination in C l i n i c a l

257

Use

E.R. Werner, D. Fuchs, A. Hausen, G. Reibnegger, H. Wächter, J . F . K . Huber . 263 Diurnal V a r i a t i o n in U r i n a r y P t e r i n E x c r e t i o n in Man A.E. Pheasant

267

The R e l a t i o n s h i p between P t e r i d i n e s and Colour Dimorphism in the Scorpion F l y M. Tsusue, M. Nakagoshi

271

P t e r i d i n e B i o s y n t h e s i s and M a n i f e s t a t i o n of Pigment Phenotypes in Normal and N e o p l a s t i c C e l l s of G o l d f i s h in V i t r o M. Masada, J . Matsumoto, M. Akino

275

L i g h t Depending Regulations of P t e r i d i n e s in the Retina G. C r e m e r - B a r t e l s , K. Krause D i f f e r e n c e s in the Regulation of T e t r a h y d r o b i o p t e r i n B i o s y n t h e s i s Neuronal C e l l s in Culture

279 in

J.H. Woolf, C.A. N i c h o l , D.S. Duch

283

NADPH- and NADH-Specific D i h y d r o p t e r i d i n e Reductase in Teratocarcinoma C e l l s i n C u l t u r e : E f f e c t of Nerve Growth Factor Treatment of the C e l l s on the Enzyme A c t i v i t i e s N. N a k a n i s h i , K. Ozawa, S. Yamada, K. A k i b a , N. Sato

287

R e v e r s i b i l i t y of ot-Diketo Reductase A c t i v i t y by S e p i a p t e r i n Reductase S. Katoh, T. Sueoka, S. Yamada

291

Non-Enzymatic S y n t h e s i s of " D r o s o p t e r i n s " from D i h y d r o p t e r i n and 2-Amino4 - 0 x o - 6 - A c e t y l - 3 H , 9H-7,8-Dihydropyrimido [ 4 , 5 b ] [1 ,4]Diazepine D.R. Paton, G.M. Brown

295

XIV BH4 B i o s y n t h e s i s : ^H-NMR Evidence f o r the P y r u v o y l - T e t r a h y d r o p t e r i n Synthase C a t a l y z e d Formation of 6 - P y r u v o y l - T e t r a h y d r o p t e r i n from Dihydroneopterin Triphosphate S . G h i s l a , S. Takikawa, P. S t e i n e r s t a u c h , T. H a s l e r , H.-C. C u r t i u s

299

C a t a b o l i s m of T e t r a h y d r o b i o p t e r i n in Man A. N i e d e r w i e s e r , A. M a t a s o v i c , T. K ü s t e r , W. Staudenmann, W. P f l e i d e r e r , S. S c h e i b e n r e i t e r Pteridine Biosynthesis

in Human Amniocytes and C h o r i o n i c

Villi

J. F e r r e , E.W. N a y l o r

309

The Role of the Kidney in B i o p t e r i n

Metabolism

J . - L . Dhondt, J.M. Hayte, C. N o e l , M. Dracon, G. L e l i e v r e , A. Tacquet Tetrahydrobiopterin-Producing and Man

305

Enzyme A c t i v i t i e s

315

in L i v e r of Animals

T. H a s l e r , A. N i e d e r w i e s e r

319

E f f e c t of 3-Hydroxykynurenine on GTP C y c l o h y d r o l a s e A c t i v i t y and on the P t e r i d i n e P a t t e r n i n D r o s o p h i l a melanogaster F . J . S i l v a , J . L . Mensua

323

T e t r a h y d r o b i o p t e r i n Metabolism in Normal B r a i n , S e n i l e Dementia o f the Alzheimer Type and Down's Syndrome J.M. Anderson, C.G.B. Hamon, R.A. Armstrong, J . A . B l a i r Summary: B i o l o g y of

327

Pteridines

M.A. Parniak

331

B i o s y n t h e s i s of P t e r i d i n e s and Metabolism of Aromatic Amino A c i d s D r o s o p h i l a melanogaster

in

Y. B e l , J . F e r r e The I n h i b i t i o n of tRNA T r a n s g l y c o s y l a s e s M e t a b o l i c Control of Gene E x p r e s s i o n

335 by P t e r i d i n e s as a Mean f o r

H. K e r s t e n , H.J. A s c h h o f f , P. Böhm, E. Zubrod

339

Modulation of Dopamine S y n t h e s i s by Nerve Terminal A u t o r e c e p t o r s : A Role for Tetrahydrobiopterin? M.P. Galloway, R.A. Levine

347

XV A Monoclonal Antibody Reacting with All Three Aromatic Amino Acid Hydroxylases R.G.H. Cotton, I.G. Jennings, R. Kuskinsky, C.W. Chow, E. Haan, W.J. McAdam, C. Bell, F. Morgan, H. Nakata, A.C. Cuello

Alterations in Cofactor-Dependent Activity of Phenylalanine as a Function of pH

351

Hydroxylase

M.A. Parniak, S. Kaufman

355

Interaction of a Monoclonal Antibody with Rat Liver Hydroxylase

Phenylalanine

M.A. Parniak, I.G. Jennings, R.G.H. Cotton

The Effect of Dietary Iron on the Activity of Rat Liver Hydroxylase

359

Phenylalanine

M.D. Davis, S. Kaufman

363

Strict Requirement of F e 2 + for Tryptophan 5-Monooxygenase from Mouse Mastocytoma, P-815 H. Hasegawa, A. Ichiyama

369

Tetrahydrobiopterin and the Regulation of Catecholamine Synthesis in Bovine Adrenal Medullary Chromaffin Cells R.A. Levine, K.L. Keiner

373

Cofactor Activity of Tetrahydropterins for Tyrosine Hydroxylation in PC12 Pheochromocytoma Cells D.S. Duch, J.F. Reinhard, M.P. Edelstein, J.Y. Chao, C.A. Nichol

Summary.: Pterin-Dependent Biological

377

Reactions

R.A. Levine

Tetrahydrobiopterin Deficiency. Analysis from an International

381

Survey

J.L. Dhondt

385

Cofactor Specificity of 6,6-Disubstituted Tetrahydropterins, and their Potential in the Treatment of Neurological Disorders J.E. Ayling, S.W. Bailey, S.B. Dillard

Normal Concentrations of Tetrahydrobiopterin in the Cerebrospinal of Patients with Dihydropteridine Reductase Deficiency K. Hyland, I. Smith, D.W. Howells

391

Fluid 395

XVI Prenatal Diagnosis of Tetrahydrobiopterin Deficiency A. Niederwieser, H. Shintaku, T. Hasler, H.-C. C u r t i u s , H. Lehmann, D. Leupold, 0. Guardamagna, A. Ponzone, H. Schmidt

399

Defective Biopterin Biosynthesis in a Chinese Infant C.R. S c r i v e r , C.L. Clow, P. Kaplan, G.V. Watters, C. Laberge, F. Mohyuddin, S. M i l s t i e n

403

Correlation of Dihydropterine Reductase Cross Reacting Material with Non-Responsiveness to a Tetrahydrobiopterin Load R.G.H. Cotton, I.G. Jennings, G. Bracco, A. Ponzone, 0. Guardamagna

407

Neopterin and Dihydroneopterins in Serum of Controls and Patients with Various Diseases. Circadian Rhythm of Neopterin. Influence of C o r t i s o l ? H. Rokos, H. F r i s i u s , R. Kunze

411

Neopterin Content and GTP-Cyclohydrolase A c t i v i t y of Blood C e l l s of Controls and Patients P o s i t i v e for LAV/HTLV-III Antibodies K. Rokos, R. Kunze, W. Lange, M.A. Koch

415

Neopterin in Bone Marrow Transplant Monitoring: An Improvement in i t s Use for GVHD Diagnosis P. Mura, A. P i r i o u , D. R e i s s , F. Guilhot, E. Benz-Lemoine, J. Tanzer

419

Importance of Neopterin Determination in I n d i v i d u a l s at Risk for AIDS D. Fuchs, A. Hausen, G. Reibnegger, E.R. Werner, H. Wächter, H.G. Blecha, H. Roessler, B. Unterweger, H. Hinterhuber, P. Hengster, T. Schulz, M.P. D i e r i c h , D. Renner

427

Biochemical Aspects of Pteridines During Interaction Between Activated T-Lymphocytes and Monocytes/Macrophages in Patients B. Andondonskaja-Renz, H.J. Z e i t l e r

431

Summary: Pterins and Human I l l n e s s D. Fuchs, H. Wächter

443

XVII

S E C T I O N D: F O L A T E S AND P T E R I N S

IN M A M M A L I A N

TISSUES

F o l a t e s in T i s s u e s and C e l l s . S u p p o r t for a " T w o - T i e r " H y p o t h e s i s R e g u l a t i o n of O n e - C a r b o n M e t a b o l i s m

of

C.L. K r u m d i e c k , I. Eto

447

E l e v a t i o n in the Rate of C e l l u l a r F o l a t e C a t a b o l i s m in the Rat

in M i d - P r e g n a n c y

J.M. S c o t t , E. W i l s o n , D.G. Weir

467

T h e Red B l o o d Cell as a S t o r a g e Site for Folic

Acid

S.E. S t e i n b e r g

471

in B l o o d F o l l o w i n g Low Dose

M e t h o t r e x a t e a n d B i o p t e r i n Levels A d m i n i s t r a t i o n for P s o r i a s i s

R.J. L e e m i n g , R.M. C o n l o n , A. P o l l o c k , J. Kumari

Summary: Folates

475

in T i s s u e and Cells

D.G. P r i e s t

479

F o l a t e R e s c u e of N 2 0 - I n d u c e d

Inhibition

in M u r i n e

Lymphocytes

J.G. H i l t o n , B.A. C o o p e r , W. Lapp

F o l a t e A b s o r p t i o n and M e t a b o l i s m

481

in Alcohol

Fed Rats

J.M. N o r o n h a , V. Kesavan

C h r o n i c a n d A c u t e Effects of Ethanol E x c r e t i o n of F o l a t e in Rats

485

on Renal

Clearance and Urinary

K.E. M c M a r t i n , B.H. E i s e n g a , T . D . C o l l i n s , L. B a i r n s f a t h e r

489

E f f e c t of C h r o n i c A n t i c o n v u l s a n t T r e a t m e n t on F o l a t e C o n c e n t r a t i o n s the Rat

in

G.F. Carl

Alcohol

495

Induced Methyl

G r o u p W a s t a g e as a P o s s i b l e C a u s e of Fatty

A . M . M o l l o y , J.M. S c o t t , D.G. W e i r

Folate Catabolism D. A l - H a d d a d , A.E.

in the S y r i a n G o l d e n

Liver 505

Hamster

P h e a s a n t , J.A. B l a i r , C . G . B . H a m o n

509

XVIII The Level o f F o l a t e C a t a b o l i s m i n Normal Human P o p u l a t i o n s , t h a t the C u r r e n t Level o f RDA f o r F o l a t e i s E x c e s s i v e

Suggesting

J . M . M c P a r t l i n , D.G. W e i r , G. C o u r t n e y , H. M c N u l t y , J . M . S c o t t

M i c r o d e t e r m i n a t i o n of F o l a t e Monoglutamates i n Serum by L i q u i d Chromatography w i t h E l e c t r o c h e m i c a l D e t e c t i o n

513

High-Performance

K. I w a i , K. I n o u e , M. Kohashi

Folate Status in P s y c h i a t r i c mitter Metabolites T. B o t t i g l i e r i , E.H. R e y n o l d s

517

Patients:

R e l a t i o n s h i p w i t h CSF N e u r o t r a n s -

M. L a u n d y , M.W.P. C a r n e y , T . K . N . C h a r y , B . K .

Toone, 523

F o l a t e , T h i a m i n e and V i t a m i n B12 L e v e l s

in Outpatient

Epileptics

M . I . Botez

E f f e c t of A s p i r i n Liver Bioassay

527

I n g e s t i o n on F o l a t e B i o a v a i l a b i l i t y

E v a l u a t e d by Rat

K. Hoppner, B. Lampi

531

B r u s h B o r d e r P t e r o y l p o l y g l u t a m a t e H y d r o l a s e i n P i g Jejunum C . J . C h a n d l e r , T . T . Y . Wang, C.H. H a l s t e d

539

Use o f C I - 9 2 0 i n the C h a r a c t e r i z a t i o n o f the M e t h o t r e x a t e D e f e c t in a R e s i s t a n t Human Leukemic CCRF-CEM C e l l L i n e

Transport

G. J a n s e n , J . H . S c h o r n a g e l , G. R i j k s e n , J . de G i e r

543

The E f f e c t o f O x i d a t i v e S t r e s s on M e t h o t r e x a t e (MTX) T r a n s p o r t by Rat Hepatocytes M . S . Rhee, A. P u p o n s , Z. Nimec, J . G a l i v a n

F o l a t e T r a n s p o r t by P i g

Intestinal

547

Brush Border

Vesicles

A.M. R e i s e n a u e r

Depressed Folate Transfer Across D e f i c i e n c y i n the Rat

551

the Mammary Gland Secondary to

Iron

D . L . O ' C o n n o r , M.F. P i c c i a n o , A . R . Sherman, S . L . B ü r g e r t

S t u d i e s on the Mechanism o f F o l a t e T r a n s p o r t D.W. H o m e

in I s o l a t e d

555

Hepatocytes 559

XIX A n a l y s i s of the pH and Na+ Dependence of I n t e s t i n a l Folate Transport I . H . Rosenberg, J. Zimmermann, J. Selhub

563

I n t e s t i n a l Transport of Folate. Characterization of the Transport Events at the Brush Border and the Basolateral Membranes H.M. S a i d , W.B. Strum

567

The Synthesis of Some Novel Probes of Folate Transport and Binding R.J. Kempton, L.A. Sams, K.M. Harpring, A.G. Smith, F. Kohrs, E. Price, J.H. Freisheim

571

P h o t o a f f i n i t y Probes of Dihydrofolate Reductase and of the Membrane C a r r i e r f o r Folate Analogues E.M. Price, P.L. Smith, J.H. Freisheim

575

The Use of Biosynthetic Radioactive Folylpolyglutamates to Study I n t e s t i n a l Digestion and Absorption of Folate B. D a r c y - V r i l I o n , J. Selhub, I.H. Rosenberg

579

Selective I n h i b i t i o n of Bacterial Carboxypeptidase G and Pancreatic Conjugase by 2-Mercaptomethylglutaric Acid T . I . Kalman, V.K. Nayak, A.R.V. Reddy

583

Summary: Folate Absorption and Transport I.H. Rosenberg

587

Studies on Glycine-N-Methyltransferase C. Wagner, R.J. Cook Folate Binding Protein from Pediococcus Cerevisiae S t r a i n s Active Transport Systems for Folates

593 Possessing

F. Mandelbaum-Shavit

597

Immobilized Folate Binding Protein from Cow's Milk Used for Quantitation of Folate S . I . Hansen, E. Nextf, J. Holm

603

Characterization of Rabbit Antibodies Against the Folate Binding Protein from Cow's Milk M. Hoier-Madsen, S . I . Hansen, J. Holm

607

XX SECTION E: ENZYMOLOGY OF FOLATES 1. Thymidylate Synthase

Genetic and Chemical Studies on Thymidylate Synthase F. Maley, F.K. Chu, D.K. West, G.F. Maley

613

Active Site Probes of Thymidylate Synthetase A.D. Broom, I.Y. Vang

631

Resonance-Raman Spectroscopic Identification of the Transient Intermediates Formed in the Native Ternary Complex with Thymidylate Synthase A.L. Fitzhugh, S. Kaufman, S. Fodor, T.G. Spiro

639

The Reversal of the Cytotoxicity of Folate-Based Thymidylate Inhibitors in Cultures LI210 Cells

Synthase

A.L. Jackman, A.H. Calvert, R.G. Moran

Thymidylate Synthase Activity in the Tapeworm, Hymenolepis

645

diminuta

J. Ciesla, Z. Zielinski, W. Rode, B. Machnicka

651

Identification of Poly G Bound to Thymidylate Synthase J. Thorndike, R.L. Kisliuk

Methotrexate Derivates of Deoxyuridylate Showing Inhibitory for Thymidylate Synthetase from Lactobaci11 us casei

655

Properties

S. Webber, R. Nazarbaghi, J.M. Whiteley

Comparison of Thymidylate Synthase Isolated from Mouse Normal Tumour Tissues

659

and

Z. Zielinski, J. Ciesla, B. Kedzierska, W. Rode

663

Thymidylate Synthase Interaction with Analogues of dUMP and 5-Fluorid2'-Deoxyuridine-5'-Phosphate with Modified Phosphate Groups W. Rode, B. Kedzierska, T. Kulikowski, D. Shugar

667

An In Vitro System for Studies on Inhibition of the Thymidylate Cycle H. Rebandel , Y. Gaumont, R.L. Kisliuk

671

XXI N ^ O - P r o p a r g y l - 5 , 8 - D i d e a z a f o l i c A c i d Polyglutamates as I n h i b i t o r s o f Thymidylate Synthase and t h e i r I n t r a c e l l u l a r Formation E. S i k o r a , A . L . Jackman, D.R. Newell, K.R. Harrap, A.H. C a l v e r t , T.R. J o n e s , K. Pawelczak, B. R z e s z o t a r s k a S e q u e n t i a l CMF i n M e t a s t a t i c B r e a s t Cancer: An Attempt to E f f e c t i v e n e s s by Means of Biochemical Modulation

675

Increase

P. Pronzato, L. Repetto, D. Amoroso, A. A r d i z z o n i , G. B e r t e l l i , P.F. Conte, V. Fusco, M. G u l i s a n o , R. Rosso

681

Model S t u d i e s of Thymidylate Synthase Reaction P . F . C . Van der M e i j , E. H i l h o r s t , T . B . R . A . Chen, E.R. de Waard, U.K. P a n d i t Synergistic

687

I n t e r a c t i o n Between F o l i n i c A c i d and the F 1 u o r o p y r i m i d i n e s

K. Keyomarsi, R.G. Moran

2. M e t h i o n i n e Methionine

691

Biosynthesis

Biosynthesis

R.G. Matthews, D.A. J e n c k s , V. F r a s c a , K.D. Matthews Impaired Formylation and Uptake of T e t r a h y d r o f o l a t e by Rat Small F o l l o w i n g Cobalamin I n a c t i v a t i o n

697 Gut

J . P e r r y , R. Deacon, M. Lumb, I . Chanarin Heterogeneity i n F u n c t i o n a l Methionine Synthase

709 Deficiency

D. W a t k i n s , D . S . R o s e n b l a t t Summary: Methionine

Biosynthesis

E.L.R. Stokstad

3. F o l y l

713

Polyglutamate

Folylpoly-y-glutamate

717

Synthetase Synthetase

B. Shane

719

C y t o t o x i c E f f e c t s and I n h i b i t i o n of F o l y l p o l y g l u t a m a t e Synthetase by F o l a t e Analogs C o n t a i n i n g 2 , Omega-Diaminoalkanoic A c i d s J . J . McGuire, J . R . P i p e r

729

XXII An Upstream Gene R e g u l a t e s the E x p r e s s i o n o f F o l y l p o l y g l u t a m a t e Synthetase-Dihydrofolate Synthetase in Escherichia coli A. B o g n a r , C. O s b o r n e , B. Shane

733

Occurence and S y n t h e s i s of P t e r o y l - | f - g l u t a m y l - t f - g l u t a m y l - p o l y - o t glutamates in E s c h e r i c h i a c o l i R. F e r o n e , M. H a n l o n , S . S i n g e r , D. Hunt

Summary: F o l y l p o l y g l u t a m a t e

737

Synthesis

E. C o s s i n s

741

The M e t a b o l i s m of

Pteroylpolyglutamates

R.L. K i s l i u k

Dihydropteroyl

743

Hexaglutamate and T4 Phage B a s e p l a t e Assembly

B. S z e w c z y k , K. S z e w c z y k , L . M . K o z l o f f

757

E f f e c t s o f P o l y g l u t a m y l a t i o n on F o l a t e C o f a c t o r and A n t i f o l a t e A c t i v i t y i n the T h y m i d y l a t e S y n t h a s e C y c l e o f P e r m e a b i 1 i z e d M u r i n e Leukemia L 1210 C e l l s T.I.

Kaiman

763

Summary: P t e r o y l p o l y g l u t a m a t e

metabolism

R . E . Mackenzie

4. D i h y d r o f o l a t e

Recent Advances

767

Reductase

in the Study of D i h y d r o f o l a t e

Reductase

R . L . B l a k l e y , J . R . Appleman

The 2 . 8 8 S t r u c t u r e o f a Type I I

769

P l a s m i d Encoded D i h y d r o f o l a t e

D.A. M a t t h e w s , S . L . S m i t h , D . P . B a c c a n a r i , J . J . B u r c h a l l , S . J . J. Kraut

Mutations B.I.

i n the Human D i h y d r o f o l a t e

Reductase Oatley, 789

Reductase

S c h w e i t z e r , S . S r i m a t k a n d a d a , S . K . Dube, J . R . B e r t i n o

T h e o r e t i c a l S t u d i e s o f the S t r u c t u r e , C o n f o r m a t i o n a l p e r t i e s of A n t i c a n c e r F o l a t e I n h i b i t o r s W.J. Welsh, V. Cody

and E l e c t r o n i c

793

Pro799

XXIII B i n d i n g of I n h i b i t o r s w i t h S p i n - L a b e l e d S i d e C h a i n s to Reductase (DHFR) from S e v e r a l S p e c i e s

Dihydrofolate

R . L . B l a k l e y , R . L . K u l i n s k i , J . R . Appleman, J . R . P i p e r

803

A f f i n i t y L a b e l i n g o f D i h y d r o f o l a t e Reductase w i t h an I o d o a c e t y l Analogue of Methotrexate

Lysine

T . J . Del camp, A. R o s o w s k y , P . L . S m i t h , J . E . W r i g h t , J . H . F r e i s h e i m

Escherichia coli

807

D i h y d r o f o l a t e Reductase I s o l a t e d as a F o l a t e Complex

S . S . Joyner, D.P. Baccanari

811

D i h y d r o f o l a t e Reductase from Soybean

Seedlings

S . Ratnam, T . J . Delcamp, J . H . F r e i s h e i m

Use o f A n t i b o d i e s as S t r u c t u r a l and E n z y m e - L i g a n d Complexes

815

Probes o f D i h y d r o f o l a t e

Reductases

M. Ratnam, T . J . Delcamp, J . H . F r e i s h e i m

819

C o o p e r a t i v i t y i n I n h i b i t o r B i n d i n g to N e i s s e r i a f o l a t e Reductase

gonorrhoeae

Dihydro-

R.L. T a n s i k , D.P. Baccanari

823

pH S t u d i e s o f the R e a c t i o n C a t a l y z e d by D i h y d r o f o l a t e Reductase from E. c o l i J . F . M o r r i s o n , S . R . Stone

827

E f f e c t s o f pH on R e a c t i o n s C a t a l y z e d by D i h y d r o f o l a t e Reductase from Chicken L i v e r J . F . M o r r i s o n , S . R . Stone

Unconjugated D i h y d r o p t e r i n S u b s t r a t e s Reductase

831

f o r Bovine L i v e r

Dihydrofolate

G.K. S m i t h , S . D . B a n k s , E . C . Bigham, C . A . N i c h o l

Enzymatic C h a r a c t e r i z a t i o n o f Recombinant Human R e d u c t a s e Produced i n coli D. S t u b e r , H. B u j a r d , E. H o c h u l i , H.P. K o c h e r , E . K . W e i b e l , F. W i n k l e r , R . L . Then

835

Dihydrofolate I . Kompis, K. Talmadge,

L e v e l s o f F o l a t e s and M e t h o t r e x a t e P o l y g l u t a m a t e F o r m a t i o n i n Hamster Ovary C e l l s L a c k i n g D i h y d r o f o l a t e Reductase P. Joannon, H. G o l d b e r g , V.M. Whitehead, D . S . R o s e n b l a t t , M . J . D. B e a u l i e u

839

Chinese Vuchich, 843

XXIV Methotrexate in Adjuvant

Arthritis

J . G a l i v a n , M.C. Rehder, S . Kerwar

847

Computer G r a p h i c M o d e l i n g i n Drug D e s i g n : C o n f o r m a t i o n a l A c t i v e - S i t e M o d e l i n g of L i p o p h i l i c D i a m i n o p y r i m i d i n e s

Analysis

and

V. Cody

851

E f f e c t o f A n t i f o l a t e s 1 0 - M e t h y l - and on a Human B r e a s t Cancer C e l l L i n e

1O-Ethyl-10-Deaza-Aminopterin

F. M a n d e l b a u m - S h a v i t

855

B i o c h e m i c a l and C y t o t o x i c E f f e c t s of the E r y t h r o - and T h r e o - I s o m e r s Gamma-Fluoro-Methotrexate

of

J . J . M c G u i r e , J . G a l i v a n , J . K . Coward

5. Other F o l a t e

861

Enzymes

C i - T e t r a h y d r o f o l a t e - S y n t h a s e , a M u l t i f u n c t i o n a l Enzyme I n v o l v e d P u r i n e M e t a b o l i s m , and i t s R e l a t e d M o n o f u n c t i o n a l Enzymes

in

K.W. Shannon, T . R . W h i t e h e a d , C. S t ä b e n , J . C . R a b i n o w i t z

The I n t e r a c t i o n o f T e t r a h y d r o p t e r o y l p o l y g l u t a m a t e s Hydrofolate Synthetase

with

865

1O-Formyltetra-

V. S c h i r c h , B. S t r o n g , G. J o s h i , R. L u r a , T. D e n n i s

Summary: F o l a t e s

887

in C 1 Metabolism

V. S c h i r c h

891

Chemical A s p e c t s o f D i m e t h y l g l y c i n e Dehydrogenase and Dehydrogenase

Sarcosine

R . J . C o o k , P . G e t t i n s , C. Wagner

Folate Normalizes

E l e v a t e d PRPP L e v e l s of F o l a t e - D e f i c i e n t

893

HL-60

Cells

J . G h i t i s , C. S c h r e i b e r , S . Waxman S t e r e o c h e m i s t r y o f H y d r i d e T r a n s f e r to NADP + by M e t h y l e n e f o l a t e Dehydrogenase from P i g L i v e r J . Green, R.G. Matthews, R . E . MacKenzie

897

Tetrahydro901

XXV The Effects of Thyroxine Status on Hepatic Levels of tetrahydrofolic Acid: NADP Oxidoreductase

10-Formyl-

J.M. Keating, H. Choe, E.L.R. Stokstad

905

Interaction of Homocysteine with Methionine

Synthetase

G.P. Lewis

909

Minor Form of Human Hepatic Betaine: Homocysteine

S-Methyltransferase

W.E. Skiba, M.S. Wells, J.H. Mangum, W.M. Awad

913

Studies of Cobalamin-Dependent Methionine Synthase from Escherichia coli B V. Frasca, W.R. Dunham, R.H. Sands, B.S. Riazzi , R.G. Matthews

Inhibition of Methylenetetrahydrofolate

Reductase by Adenosyl

917

Methionine

D.A. Jencks, R.G. Matthews

921

Regulation of Folate Homeostasis C. Osborne, K. Lowe, B. Shane, D.J. Cichowicz, D. Sussman, G. Milman

An Endogenous Inhibitor of Neurospora Folylpolyglutamate

925

Synthetase

E.A. Cossins, P.Y. Chan

929

Folylpolyglutamate Hydrolase from Beef Liver P.J. Vickers, R. Di Cecco, Z.B. Pristupa, K.G. Scrimgeour

933

Use of Mammalian Cell Transformation to Isolate Genomic Sequences Encoding Human Fo.lyl polygl utamate Synthetase (FPGS) and Dihydrofolate Reductase (DHFR) S.M. Taylor, N. Davidson, R.G.Moran

937

SECTION F: METHOTREXATE AND OTHER ANTI-FOLATES

Antifolates: Expanding Horizons in 1986 B.A. Chabner, C.J. Allegra, J. Baram ..

945

XXVI Deaza D e r i v a t i v e s of T e t r a h y d r o f o l i c A c i d . A New C l a s s of F o l a t e Antimetabolite G.P. B e a r d s l e y , E.C. T a y l o r , G.B. G r i n d e y , R.G. Moran S t r u c t u r e A c t i v i t y R e l a t i o n s h i p s of Novel T r i a z i n e

953

Antifolates

C. D i a s - S e l a s s i e , Y.C. Zheng, H. Zhu, C. Hansch, T. Khwaja, J.H. F r e i s h e i m , T . J . Delcamp

959

The E f f e c t of 7-Hydroxymethotrexate on the Antitumor A c t i v i t y and Host T o x i c i t y of Methotrexate In Vivo P.A. Newton, R.A. F i n c h , T . L . Avery

953

Summary: The B i o l o g y of A n t i f o l a t e s J. Gallivan C r y s t a l S t r u c t u r e of Methotrexate and i t s Conformational with Related S t r u c t u r e s

967 Comparison

P.A. S u t t o n , V. Cody E f f e c t o f Methotrexate on Reduced F o l a t e s and Thymidylate During Growth of L1210 C e l l s

969 Synthesis

D.G. P r i e s t , V. Kesavan, M.T.Doig D i h y d r o f o l a t e Reductase from Human Osteosarcoma C e l l s R e s i s t a n t Methotrexate

973 to

J . K . S a t o , T . J . De-Frank

977

Evidence f o r D i r e c t I n h i b i t i o n of M e t a b o l i c Pathways as a Mechanism of A c t i o n of Methotrexate C . J . A l l e g r a , J . Baram, B.A. Chabner I n h i b i t i o n of D i h y d r o f o l a t e Reductase and Thymidylate Synthase by Methotrexate Polyglutamate Analogues L a c k i n g " I n t e r n a l " « - C a r b o x y l

981

Groups

A. Rosowsky, R.A. F o r s c h , M.M. Wick, J.H. F r e i s h e i m , P.V. Danenberg, T . I . Kalman

985

The A n t i f o l a t e A c t i v i t y o f Poly-jf-Glutamyl D e r i v a t i v e s of Methotrexate, 10-Deazaaminopterin and 1 0 - E t h y l - 1 0 Deazaaminopterin R . L . K i s l i u k , Y. Gaumont, P. Kumar, M.G. N a i r , B.T. Kaufman

989

XXVII P o l y g l u t a m a t i o n of the Thymidylate Synthase I n h i b i t o r , N ^ O - P r o p a r g y l 5 , 8 - D i d e a z a f o l i c A c i d (CB 3 7 1 7 ) , in Organs of E h r l i c h A s c i t e s Carcinoma-Bearing Mice M. Manteuffel-Cymborowska, B. Kaminska, B. G r z e l a k o w s k a - S z t a b e r t

5 , 8 - D i d e a z a f o l a t e s as S u b s t r a t e s f o r F o l y p o l y - # - G l u t a m a t e from Pig L i v e r

Synthetase

J . B . Hynes, D.J. C i c h o w i c z , B. Shane 5 - S u b s t i t u t e d - 5 - D e a z a Analogues of C l a s s i c a l

993

997 Antifolates

J . R . P i p e r , G.S. McCaleb, J . A . Montgomery, F.M. S i r o t n a k

1001

A z i d o - S u b s t i t u t e d A n t i f o l a t e Drugs: S y n t h e s i s , S t r u c t u r e , and A c t i v i t y P.K. B r y a n t , K.P. Wong, J . C o l b y , C.H. Schwalbe, M.F.G. R . J . G r i f f i n , E.A. B l i s s Summary: Novel

Stevens, 1005

Antifolates

R. Jackson

1009

AUTHOR INDEX

1011

SUBJECT INDEX

1017

L I S T OF PARTICIPANTS

1033

HOW IT ALL BEGAN: A BRIEF HISTORY OF THE PTERIDINE SYMPOSIA

Adrien Albert Chemistry (Science), the Australian National University, Canberra, Act 2601, Australia

Introduction Who could have seen that, from their modest beginning in Paris little more than thirty years ago, the Pteridine Symposia would grow into such a well-established series, steadily expanding in interests and attendance?

It was in 1951, I remember, that I paid my first visit to Professor Michel Polonovski's Department of Biological Chemistry in Paris.

It was in the Faculty of Medicine, just off the east

end of the Boulevard de Saint Germain.

I had recently been

appointed to the Chair of Medical Chemistry in the newly-formed Australian National University.

However that federal

institution was only at the stage of drawing plans for the buildings.

Meanwhile, they funded me to form an Australian

research group in London, in the Wellcome Research Institution.

When we met, Polonovski and I eagerly exchanged details of our researches in pteridine chemistry, a subject not widely practised at that time, but one that fired both of us with enthusiasm.

As we spoke of it, we began to wish for an

Occasion, where all who were interested in pteridine-based science could meet to exchange information and stimulate one

Chemistry and Biology of Pteridines 1986 © 1986 Walter d e Gruyter & Co., Berlin • New York - Printed in Germany

2

another.

Polonovski thought that, provided its orbit was

confined to Western Europe, he could just about stretch some funds to bring it off!

Later he wrote to Des Brown and myself

in London: 'Le sujet du symposium sera: Etudes sur les ptérines: répartition, constitution, analyse, synthèse, propriétés physicochimiques, ptérines naturelles, action physiologique et pharmacodynamique'.

The First Symposium And so it happened that, September 28-30 1952, the First Symposium on the Chemistry and Biology of Pteridines took place in the Paris Medical Faculty, in drizzly overcast autumn weather.

The meeting attracted 18 listeners.

Our lectures were

published only individually, as papers.

Talks were given by Polonovski and his collaborators (R.-G. Busnel and M. Pesson), by W. Jacobson of the Strangeways Research Laboratory in Cambridge, by F. Korte of the Chemisches Staats Institut in Hamburg, by F. Egami from Nagoya University (he was visiting his old University in Strasbourg at the time), and by D.J. Brown and myself.

Polonovski's group spoke of their

experiences with Isay's general synthetic method, of the necessity for modifications of it to get reproducible high yields, and of some mystifying by-products obtained when preparing 4,5-diaminopyrimidines, those necessary starting materials.

Korte spoke of the naturally-occurring pteridines

whose constitutions he and R. Tschesche were beginning to

unravel.

Egami acquainted us w i t h the surprising amount of work

done in Japan on the paper-chromatographic isolation of pteridines from silkworms and fish.

Jacobson struck a clinical

note by identifying the stage of human mitosis inhibited by methotrexate, a folic acid analog recently introduced for treating leukemia in children.

I drew attention to the huge and

unprecedented electronic gradient existing in the highly n-deficient (my newly-coined word!) pteridine nucleus.

Brown

and I told how we practised the study of mono-substituted pteridines as an essential first step in assessing how this 'electron-thirst' could be attenuated, stepwise, by inserting electron-releasing substituents

(such as -NH2 or =0).

This

strategy revealed the rules governing solubility and stability in the whole pteridine series.

Photo 1, taken by Prof. Egami at the time, shows (in the back row, left to right): Drs. Jacobson, Busnel, Korte, Brown and (a little to the fore) Prof. Polonovski; also (in the front row) Madame Polonovski, myself, and Madame Busnel.

Our hosts' kind hospitality provided us with some memorable meals.

But, one lunchtime, w i t h a merry twinkle in his eye,

Michel conducted us to a typical Left-Bank establishment: la Grenouille'.

'Roger

This, as y o u may have guessed, served only one

kind of dish, one whose amphibian delicacy drew varied comments.

4

Photo 1

The Second Symposium Back in England, Jacobson and I, greatly stimulated by the Paris meeting, began to wish for a larger one, with enough funding for us to be able to invite American colleagues.

When approached,

the Ciba Foundation readily agreed to our suggestion. Accordingly, the Second Pteridine Symposium took place in the Foundation's London premises in Portland Place (March 22-26, 1954).

Spring had arrived early that year, and the dull,

war-scarred Squares of London sprang to life around drifts of golden daffodils blooming under pink-flowering Prunus trees.

5

This provided a welcoming note for the Symposium's American visitors, and it turned out to be a good omen for our Meeting.

There were 29 participants: Dr. Ruth Bellairs, F. Bergel, D.J. Brown, R.-G. Busnel, Marie Coates, H.O.J. Collier, J. Colsky, Donna Cosulich, J. Druey, Gertrude Elion, H.S. Forrest, R.H. Girdwood, A. Haddow, G.H. Hitchings, Dorris Hutchison, G.W. Kidder, F. Korte, S.F. Mason, R.H. Nimmo-Smith, M. Polonovski, E.C. Taylor, G.M. Timmis, R. Tschesche, M. Webb, F.J. Wolf, H.C.S. Wood, D.D. Woods, as well as the two Chairmen: Dr. Jacobson and myself.

I recall opening that meeting with the thought that, sixty-five years earlier, Frederick Gowland Hopkins had isolated the first pteridine from Nature, in fact from a butterfly (1).

At the

time of this Second Symposium, the only naturally-occurring pteridine known to be essential for human metabolism was folic acid, and the program gave it due prominence in clinical, biochemical, and medical contributions.

The Ciba Foundation set

a splendid example in accelerated publication of the Proceedings which came out in the same year (2).

Photo 2 shows Dr. G.E.W. Wolstenholme (Director of the Foundation) flanked by G.M. Timmis (left), who had just told us about his new, nitroso-group-participating reaction for the unambiguous synthesis of insecurely-assigned pteridines, and (on the right) by H.O.J. Collier, who had been discussing the possibilities of pteridines in chemotherapy, a topic being developed (after some remarkable molecular simplifications) into a series

6

Photo 2

of much-used diaminopyrimidine drugs by G.H. Hitchings seen (left) with D.D. Woods in Photo 3.

Photo 4 shows M. Polonovski

(right) with F. Bergel and Photo 5 presents E.C. Taylor (right) with Donna Cosulich.

Sad to tell, Michel Polonovski lost his

life in a motoring accident shortly after this London meeting.

I recall reading a paper to that Symposium: Some Unresolved Problems in Pteridine Chemistry.

I'm happy to add that, thanks

to the stimulus of the meeting, I had solved these problems before the next Pteridine Symposium.

They turned out to have a

single solution: unexpected nucleophilic addition across a

Photo 3

highly susceptible double-bond.

Further studies of this

phenomenon kept me and my colleagues busy for many a year.

Of

these addition reactions, the most remarkable (and unexpected) was covalent hydration which, as we went on to show, affects not only pteridines but many other families of nitrogen heterocycles.

The Third Symposium Rather a long interval elapsed between this and the Third Pteridine Symposium which took place, September 1962, in Germany

Photo 4

(in Stuttgart's Technische Hochschule).

It had been arranged by

Wolfgang Pfleiderer who chaired it jointly with E.C. Taylor of Princeton. days.

There were 52 participants and it lasted for four

A historical note was struck by the invited presence of

two real pioneers of the structural chemistry of naturally-occurring pteridines: C. Schopf and R. Purrmann. Hopkins did not know the structure of the substances that he had isolated from butterflies.

The subject was not resumed until

1924 when Clemens Schopf came to study in Heinrich Wieland's Department in Munich.

They coined the name 'pterin' in 1925,

but the underlying structure remained unknown until 1940 when

Photo 5

Robert Purrmann, in the same Department, showed that xanthopterin, isoxanthopterin and leucopterin were simple derivatives of pyrimido[4,5-b]pyrazine, a known ring-system to which Wieland gave the name 'pteridine' in 1941.

Photo 6, taken from the Proceedings (3), shows us participants at this Third Symposium.

The Meeting indicated several new

lines of interest, particularly the study of biopterin whose great importance in human metabolism was beginning to be realized.

10

Photo 6

Subsequent Symposia

The Fourth Pteridine Symposium took place in 1969, in an attractive pearling village named Toba, reached from Nagoya. Our Japanese hosts had taken over a complete air-conditioned hotel for our meeting which was held in comfort, in spite of the humid external weather at the height of Japan's 'rainy season'. I speak of July 21-25.

The meeting was chaired by Prof. K.

Nakanishi and attracted about 100 'pteridinologists' (as we were beginning to call ourselves) from 7 countries. papers (4).

We presented 45

This meeting coincided with Man's first landing on

the moon, an exciting and memorable coincidence.

11

Prof. Wolfgang Pfleiderer was again host to a Pteridine Symposium when he arranged the Fifth in Konstanz (Germany), exposing us to the contemporary and colorful architecture of the new University on the shores of the Bodensee.

This took place

from the 14th to the 18th of April in 1975, in delightful Spring weather.

Seventy-one papers were read (5) to an audience of

about 150.

The Sixth Pteridine Symposium was held (September 25-28, 1978) in the Scrips Clinic and Research Foundation at La Jolla, that delightful beach resort lying just outside San Diego in the USA, near the border with Mexico.

It was attended by 230

pteridinologists who presented a total of 140 papers and posters (6).

The meetings had by now grown to a size that demanded not

an organizer so much as an organizing committee: Drs. J.J. Burchall, G.M. Brown, F.M. Huennekens, R.L. Kisliuk, S. Kaufman, J. Montgomery, T. Shiota, and J.M. Whiteley.

The Seventh Pteridine Symposium (7), so fresh in our minds, went off brilliantly during September 21-24 (1982) in St. Andrews, Scotland.

It hosted 274 participants from 24 countries, and

reflected great credit on Prof. Hamish Woods who arranged it.

The present Eighth Pteridine Symposium, in Montreal, is carrying on the established tradition.

These gatherings bring together

pteridine workers from many diverse scientific disciplines, from physical and organic chemistry, biochemistry, general biology, microbiology, and clinical medicine.

Their aim is to generate,

in enthusiasm and harmony, a cross-fertilization of ideas and

12

mutual stimulation. successful.

In this, they seem to have been very

They have provided refreshing insight into

long-standing problems and they have constantly suggested new pathways for exploration.

Long may they continue!

References 1.

Hopkins, F.G. 1889. Nature (London) 40, 335.

2.

1954. In: Chemistry and Biology of Pteridines, a Ciba Foundation Symposium. (G.E.W. Wolstenholme and M.P. Cameron, eds.). London, Churchill.

3.

1964. In: Chemistry and Biology of Pteridines. (W. Pfleiderer and E.C. Taylor, eds.). Oxford, Pergamon Press.

4.

1970. In: Chemistry and Biology of Pteridines. (K. Iwai, M. Akino, M. Goto, and Y. Iwanami, eds.). Tokyo, International Academic Printing Co. Ltd.

5.

1975. In: Chemistry and Biology of Pteridines. (W. Pfleiderer, ed.). Berlin and New York, de Gruyter.

6.

1979. In: Chemistry and Biology of Pteridines. (R.L. Kisliuk and G.M. Brown, eds.). Amsterdam, Elsevier/North-Holland.

7.

1983. In: Chemistry and Biology of Pteridines. (J.A. Blair, ed.). Berlin and New York, de Gruyter.

THE MECHANISM OF ACTION OF FOLATE AND PTERIN REQUIRING ENZYMES

S.J. Benkovic, L.J. Slieker, S.C. Daubner, L.F. Courtney, T.A. Dix, S.O. Pember, L.M. Bloom, C.A. Fierke, R.J. Mayer, J.-T. Chen, and K. Taira Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802

Introduction I am honored to follow in Dr. Frank Huenneken's footsteps and to present the Gowland Hopkins Lecture.

The previous volume in this

series began with a brief forward describing the achievements of Professor Hopkins, particularly his interest in pterin chemistry. I will attempt to highlight the advances achieved in the elucidation of the mechanism of action of enzymes that utilize tetrahydropterin or tetrahydrofolate cofactors since the Seventh International Symposium.

This will not be an exhaustive review and

will be biased towards studies conducted in my laboratory.

I am

indebted to Dr. Young whose exemplary state of the art lecture at the last Symposium provided me with a detailed frame of reference ( 1 )

•

Tetrahydrofolate Requiring Enzymes The bridged 5,10-CH2-H4folate is an active shuttle of one-carbon units at the formaldehyde level of oxidation.

A key to under-

standing the mechanism of action of enzymes that process 5,10-CH2H4folate was provided by the stereospecific chemical synthesis of (6R, 11R) -and (6R, U S ) - 5 ,10-CH2

[ 1 l-^-H, 2 H] H 4 folate and the deter-

mination of the absolute stereochemistry of the two epimers at C-ll by proton nuclear Overhauser enhancement relative to C7-H (2) . These NMR measurements in conjunction with others suggest that 5,10-CH2-H4folate adopts the saucer-shaped conformation shown for 1 (3) .

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

14

Hs !

COOH I CsH4CONHCHCHjCHJCOOH

H,N

The

1

correlation

epimers of the

of

the

stereochemical

formed

in e n z y m i c

latter's

absolute

stereochemical

ant mechanistic

implications.

A case

is the o n e

in p o i n t

thymidylate numerous enzymic

synthetase.

experiments process

supported by is n o w

studies

stereospecific

(4).

its C-ll

3

respectively

epimer

course

transfer

reaction has been both model

A reasonable alternate

stereochemical

conversion

and

H]TMP

This

involving

strengthened by

folate

carbon unit

involving

itself

assignments

to t h o s e

processes has permitted

of

the

and of

(S)

attend-

by

the subject

compounds

as w e l l

hypothetical substrates

information and

the

catalyzed

of as

and

inhibitors, from

[11-^h,H4-

H.

M

HN 1

o

H H

H

1

S

(1)

E

NHR

(2)

S

the

(R)-methyl[methyl-^H,2H,

(5).

VV u

the

pathway

derived

(6R,1IS)-5,1O-methylene

to t h e

for

elucidation

15 H RN H H,folate

NH.

H'

(3) TMP

H,

The first step in (eq.l) presumes the antiperiplanar opening of the imidazolidine ring (the lone pair of electrons on N-5 is almost antiperiplanar to the breaking C11-N10 bond). tional restraints of the fused

Since the conforma-

imidazolidine-tetrahydropyrazine

ring system preclude inversion at N5, a syn elimination would seem to be impossible.

The resulting N-iminium cation must then have

the Z configuration, as shown.

Nucleophilic attack by enzyme-

bound dUMP (eq.2) on Cll can then occur from the s_i face to give the ternary complex followed by elimination to give enzyme-bound 5-CH2-dUMP and ^ f o l a t e . The depicted conformation of the bound complex is based on l^F NMR studies performed on the abortive, stable ternary complex, FdUMP5,1O-methylenetetrahydrofolate-thymidylate native and denatured states (6,7).

synthetase, in both its

These measurements confirmed

the fluor ine and C6—H of the nucleotide to be in equatorial positions in the native enzyme complex, and the Enz-S and CII-CH2 bridge moieties to be in a trans-diaxial conformation.

Indirect

measurement of Jjjp values between C5-F and C11-2H suggested that the C5-F bond (and presumably the C5-H bond of the native species) was neither syn nor anti to the C11-N5 bond.

Thus, whatever the

conformation of the ternary complex, the enzyme must induce a conformation change prior to elimination. The formation of the methyl group has been postulated to occur through the transfer of C6-H as a hydride equivalent to the exocyclic C5-methylene of the uracil ring.

This results in oxidation

of the tetrahydropterin ring in a single two-electron step as opposed to the usual pathway for pterin oxidation through the quinonoid form in either one or two electron steps (8).

The place-

ment of the two rings in a stacked conformation within the thymidylate synthetase active site poises them for e~ transfer.

A

16 two-step 1 e~/H- radical sequence invoking the redox properties of the pterin is shown (eq.3).

This mechanism can satisfy the

observed stereochemistry provided rotational effects within the radical pair are slow relative to complete reduction.

The stereo-

chemical evidence by itself does not preclude the hydride transfer process. Our apparent understanding of the thymidylate synthetase reaction notwithstanding, the participation of 5,10-CH2~H4folate in the conversion of uridine to ribothymidine in tRNA catalyzed by the ribothymidyl synthase from S_;_ faecalis takes an unexpected course (9).

Experiments with the purified enzyme demonstrated that the

methyl group is derived from the methylene unit but that FADH2 serves as the reducing agent and not H4folate.

The hydroxymethyl-

transferases found in bacteriophages are additional examples of enzymes that utilize 5,10-CH2~H4folate, transferring the onecarbon unit as a hydroxymethyl group to form pyrimidine nucleosides' -monophosphates (4).

Whether all these reactions proceed at

least as far as a common putative exocyclic methylene intermediate and then diverge is not known. The H4folate also acts as a source of one-carbon units in reactions catalyzed by a group of enzymes referred to as methionine synthases.

There are two different classes of synthase enzymes:

those that utilize cobalamin as a prosthtetic group and those that do not (10).

Both enzymes are often found in microorganisms such

as E^ coli; mammalian tissues contain only the cobalamin-dependent form.

Both classes of synthases catalyze the transfer of a methyl

group from S-CHj-t^folate to homocysteine to form methionine. the case of the

In

coli cobalamin-dependent methionine synthase

there is now direct stereochemical data to add to other evidence that this reaction involves an intermediate methylcobalamin

(eg.4).

17 H 200 times) the

steady-state turnover rate, V^, is not affected.

In terms of the

dissociation equilibrium constant the effect is 140 fold shown).

Note, however, that k o f f

dissociation of ^ f o l a t e

(data not

( H 4 F ) — t h e rate constant for

from E--is > 300

in constrast to 1.4

s _ 1 observed for wild-type, and parallels the weak binding of 7,8-H2folate. trolling.

Thus product dissociation is no longer rate con-

Secondly, the deuterium isotope effect on Vm is 3.0,

25 in constrast to the (V)D of 1.1 for wild-type (30).

Thus the

similar turnover rates observed for the two enzymes in the steadystate measurement are coincidental with hydride transfer largely rate-limiting for the mutant enzyme. Table I. Comparison of Kinetic Parameters for Wild-type and Gly-54

K

Ma (yM)

Va (s- )

Wild-type

0. 5

12

Gly-54

140

14

a

k 0 ff (H 4 F) b (s-1)

1

1. 4

300

3. 0

Conditions: 50 mM Mes, 25 mM Tris, 25 mM ethanolamine, 100 mM NaCl, (MTEN buffer) pH 5.5, 25°C, 100 yM NADPH, enzyme and H 2 F according to K M and V values,

b Conditions: MTEN buffer, pH 7.0, 25°C. c

Kinetic deuterium isotope effect with NADPD, under same conditions as (a).

It is doubtful that the function of the Gly-54 mutant enzyme was seriously impaired by a change in conformation.

Point mutations

in general are accomodated by very minor readjustments of the protein structure and that "space" created by introducing smaller amino acids can be occupied by water molecules (31,32).

Exper-

imental evidence for the Gly-54 enzyme supports this generalization since the on and off rates for NADPH binding, the rates of DHFR coformer interconversion and their relative concentrations are in good agreement with wild-type values (33). The effect of the Leu-54 -»• Gly mutation then is both to decrease the binding interaction and to make chemical catalysis less efficient.

The primary effect of removing the isobutyl side chain

is a 140-fold, or 2.9 kcai_/mol, decrease in the free energy change obtained upon binding substrate.

However, the interaction in the

transition state for the chemical step between Leu-54 and substrate must also be important despite the remoteness of the

26 residue from the reaction center.

The difference in the rate of

hydride transfer due to the mutation is 32-fold from 450 s~l to 14 s _ 1 .

The transition state may be destablilized either by

misalignment relative to the NADPH, or by loss of a hydrophobic interaction which preferentially stabilizes the developing H4folate product.

In terms of observed free energy changes, at

saturating f^folate levels, the Leu-54 residue stabilizes the transition state for hydride transfer ca. 35-fold (AAG = 2.1 kca]_/ mol) relative to the ternary complexes.

At subsaturating H2folate

levels, one would observe both the 2.9 k c a i/mol of binding energy and 2.1 k c a i/mol of transition state stabilization.

Thus the total

stabilization by Leu-54 of the transition state for hydride transfer is ca. 5000 fold (AAG = 5.0 k c a i/mol) relative to free enzyme, assuming both mutant and wild-type have identical free energies.

With respect to methotrexate binding, K D is increased

by 300-fold or A k c a i/mol of 2.9.

It remains to be seen whether

the magnitude of these effects are general or unique to this mutation.

Acknowledgment This work was supported in part by grants to S.J. Benkovic from the National Science Foundation #DMB-8316425 A02 and the National Institutes of Health #GM24129.

References 1.

Young, D.W. 1983. (J.A. Blair, ed.).

In: Chemistry and Biology of Pteridines W. deGruyter Berlin, p. 321.

2.

Slieker, L.J. , S.J. Benkovic. 333.

3.

Poe, M., L.M. Jackman, S.J. Benkovic. 5527 .

4.

Santi, D.V., P.V. Danenberg. 1985. In: Folates and Pterins (R.L. Blakley and S.J. Benkovic, eds.). Wiley Interscience New York, Vol I. Chap. 9.

1984.

J. Am. Chem. Soc. 106, 1979.

Biochemistry 18,

27 5.

Tatum, C., J. Vederas, E. Schleicher, S.J. Benkovic, H. Floss. 1977. J. Chem. Soc. Com. Coramun., 218.

6.

Bryd, R.A., W.H. Dawson, P.D. Ellis, R.B. Dunlap. Am. Chem. Soc. 100, 7478.

7.

James, T.L., A.L. Pogolotti, K.H. Ivanetich, Y. Wataya, S.S.H. Lam, D.V. Santi. 1976. Biochem. Biophys. Res. Commun. 72, 404 .

8.

Pfleiderer, W. 1985. In: Folates and Pterins (R.L. Blakley and S.J. Benkovic, eds.). Wiley Interscience, Vol II, Chap. 2.

9.

Deik, A.S., D.P. Nagle, Jr., J.C. Rabinowitz. Chem. 255, 4387.

1978.

1980.

J.

J. Biol.

10.

Matthews, R.A. 1985. In: Folates and Pterins (R.L. Blakley and S.J. Benkovic, eds.). Wiley Interscience, Vol I, Chap. 13.

11.

Zydowsky, T.M., L.F. Courtney, V. Frasca, K. Kobayashi, H. Shimizu, L.-D. Yuen, R.G. Matthews, S.J. Benkovic, H.G. Floss. 1986. J. Am. Chem. Soc. 108, 3152.

12.

Smith, G.K., P.A. Benkovic, S.J. Benkovic. istry 20, 4034.

13.

Daubner, S.C., M. Young, R.D. Sammons, L.F. Courtney, S.J. Benkovic. 1986. Biochemistry ,25, 2951.

14.

Daubner, S.C., S.J. Benkovic. 4990.

15.

Daubner, S.C., J.L. Schrimsher, F.J. Schendel, M. Young, S. Henikoff, D. Patterson, J.A. Stubbe, S.J. Benkovic. 1985. Biochemistry 24, 7059.

16.

Lazarus, R.A., R.F. Dietrich, D.E. Wallick, S.J. Benkovic. 1981. Biochemistry 20, 6834.

17.

Lazarus, R.A., S.J. Benkovic, S. Kaufman. Chem. 258, 10960.

18.

Dix, T.A., G.E. Bollag, P. Domanico, S.J. Benkovic. Biochemistry 2_4, 2955.

19.

Wallick, D.E., L.M. Bloom, B.J. Gaffney, S.J. Benkovic. Biochemistry ¿3, 1295.

20.

Marota, J.J., R. Shiman.

21.

Bloom, L.M., B.J. Gaffney, S.J. Benkovic: (in press).

22.

Pember, S., J.J. Villafranca, S.J. Benkovic: (in press).

1984.

1985.

1981.

Biochem-

Cancer Research 45,

1983.

J. Biol. 1985. 1984.

Biochemistry _23, 1303. Biochemistry. Biochemistry.

28

23.

Storm, C.B., S. Kaufman. Coraraun. 3J2, 788.

1968.

Biochem. Biophys. Res.

24.

Dix, T.A., S.J. Benkovic.

25.

Shiman, R.: (personal communication).

26.

White, R.E., M.J. Coon.

27.

Bolin, J.T., D.J. Filman, D.A. Matthews, R.C. Hamlin, J. Kraut. 1982. J. Biol. Chem. 257, 13650.

28.

Filman, D.J., J.T. Bolin, D.A. Matthews, J. Kraut. J. Biol. Chem. 257, 13663.

29.

Stone, S.R., J.F. Morrison.

30.

Chen, J.-T.: (unpublished results).

31.

Howell, E.E., J.E. Villafranca, M.S. Warren, S.J. Octley, J. Kraut. 1986. Science 231, 1123.

32.

Chothia, C., A.M. Lesk.

33.

Cayley, P.J., S.M.J. Dunn, R.W. King. 20, 874.

Biochemistry 2±,

1985.

1980.

Ann. Rev. Bioch. 49, 315.

1984.

1985.

5839.

1982.

Biochemistry 23, 2753.

J. Mol. Biol. 182, 151. 1981.

Biochemistry

SECTION A CHEMISTRY OF PTERINS AND FOLATES

SIDE-CHAIN

CHEMISTRY

W.

Pfleiderer,

W.

Leskopf

OF

PTERIDINES

Y. K a n g ,

R. S o y k a , W. H u t z e n l a u b ,

Fakultät für C h e m i e , U n i v e r s i t ä t Konstanz U n i v e r s i t ä t s s t r a ß e 10, D - 7 7 5 0 K o n s t a n z / W .

M.

Wiesenfeldt,

Germany

Introduction Ever

since

structure

1940 w h e n of

the

R.

three

Purrmann naturally

(1-3)

xanthopterin,

isoxanthopterin , and

the

nucleus

pteridine

system dines

and

start able

a large

pterins

either

from

to f o r m

vey

of

the m o s t

done

and

Tschesche chain

bear

not

and

Glaser

that

to g i v e

in r e a s o n a b l e

approach

in t h e i r

the

directly

Goto

on t r e a t m e n t noticed

with

P^S^g.

chain

Another

on N a ? S ? 0 4 - t r e a t m e n t

of

are

the

sur-

fact

that

dealing

with

has

been

naturally their

in-

condensation

that

brominated

the C - 6 and

and

(6) a p p l i e d the

side-chain

D-erythro-neopterin

Chemistry and Biology of Reridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

side-

oxidized

I'-keto

an a n n e l a t e d of

py-

chain-1ransformation .

by c o n v e r t i n g into

an by

functional

most

1'-hydroxy

example

or

in 6 - p o s i t i o n

and Sakurai

synthesis

dibenzoyloxy-2'-oxobutyl)-side

since

through

be

pteri-

A close

chemistry

1958 a l r e a d y can

ring

surprising

various

a subsequent

in

to

approaches

is c o n c e r t e d

system

the Even

corresponding

urothion

the

side-chain

showed

yields.

that

derivative

pteridine.

little

side-chains.

(4)

pyrazine

ring

of

very

through

routes

derivative

this

in 6 - a c e t o n y l i s o x a n t h o p t e r i n

respectively

was

at

achieved

(5)

showed

pyrimidine

indicates

a carbon

indirectly

and

chemical pigments

two m a j o r

annelated

reactions

but

carbon

is u s u a l l y

The

the c o r r e s p o n d i n g

literature

nucleus,

pterins

synthetic

substitued the

modifications

in m o d i f y i n g

step

to

leucopterin

developed.

pyrazine

displacement

at the

troduction

1 ogs

substituted

common

occurring

been

the

butterfly

of a p y r a z i n e [ 2 , 3 - d ] p y r i m i d i n e ri ng

versatile

a suitably

pteridine

nucleophilic groups

have

annelation

the

of

on c o n d e n s a t i o n

appropriately rimidine

consists

number

elucidated

occurring

a

ana-

similar

6 - ( 3 ' ,4 1 thieno-ring modification (7)

lead-

32 ing

to a m i x t u r e

of 6 - s u b s t i t u t e d

involving

displacement,

reactions

respectively

pterins

and

7 ,8-dihydropterins

isomerization , reduction, of

various

N

H

hydroxy

groups

and

oxidation

(Fig.

H

1).

H

Na,SA

OH C - CH2 - CHjOH H

NEOPTERIN HN A .N

N

Intraly

and

(8,9)

sing

intermolecular

7 ,8-dihydrobiopterin at

since

thetic

varying nature

pathway

from

tetrahydrobiopterin

redox-reactions (8) a n d

pH. T h e s e

uses

have

are

however

transformations

guanosine-5 '-triphosphate (10-12)

also

been

tetrahydrobiopterin

results

analogous

H

^0H N'

HjNA , N

1

Fig.

with

OH •C - C - CHÎOH

HN'

(Fig.

not

found

respectivetoo

in the

(GTP)

to

surpri-

biosyn5,6,7,8-

2). OH OH HN

hnAt-Nx

A

XN>

H,N

A , N"

~N' H

X

H H

PHOSPHATEELIMINATING ENZYME

"

H H C-C-CHj

C-C-CH3 H H,N

N.^C-C-CH,0®3

HN

H,N A

A H

Fig. 2

H

H H

OH OH

33 The

side-chain

also

very

in 5 , 6 , 7 , 8 - t e t r a h y d r o b i o p t e r i n - 5 ' - t r i p h o s p h a t e

sensitive

reactions

giving

rise

7,8-dihydropterin change

takes

(14)

0

hydrolysis,

to the f o r m a t i o n

derivatives

place

a c i d at e l e v a t e d deri vati ve

towards

of v a r i o u s

(13). A m o r e

if D - n e o p t e r i n

temperatures (Fig.

oxidation, and

6-substituted

severe

structural

is t r e a t e d w i t h

forming

is

elimination

polyphosphorous

a pyrrolo [ 1 ,2-f]pteridine

3).

OH OH

0

900

hnVvu-^

HJI'VV'

|

M

1

W^H-^U*

W

n

n

D - NEOPTERIN

Fig. In a n a l o g y

to t r a n s f o r m a t i o n s

thro-biopterin to f o r m

has been

3

in the n u c l e o s i d e

treated with

in a s t e r e o s p e c i f i c

intermediary

ring

0

opening

N

HN' I I W ' V N r

H

cyclic

and acyl

H

' ' 0H 0H

CH,

*

acyloxonium-ion migration

o

/I OCOCH,

(15)

°

—

|

led to followed

H

I I n I t l V v i

°

NH

|

H

- c -CHS C CH,

4

via

nucleophilic

N

|

Ac

^H H

, ..o h

t H

H-Nli HjN n X - N 0 0®0

Fig.

by

derived

4).

" 3 — OCOCH,

lH

6-(L-threo-1'-acet-

have b e e n

(Fig.

?

N

IT

chloride

6-(L-threo-2 -acetoxy-1 ' -

treatment

amino-2'-hydroxy propyl)-pterin , which must an

L-ery-

1

reaction

chloropropyl)-pterin . Ammonia

chemistry

2-acetoxy-isobutyryl

A N 'H2h h n ^ - N ^ C - C - CH3

nhjj-4 ^ ^ N J LN }

H

OCOCHj

34 Addition cribed could

reactions

only

to u n s a t u r a t e d

incidentally.

be c o n v e r t e d

phenyl-ethyl) chiral

of c o m p o u n d s

reactivity

of

potential.

Other

found

such

type

interesting

and 6-keto

(17)

derivatives

studies

n

Y

C H j 0 H

H

des-

to

the

(Fig.

KMnOt/OHe

AcOH/HjO

SO,

80

Oj

have

syntherecent7,7-

6-carboxal-

5).

NO REACTION

OR HjO,

02-

-

new

of 6 - s u b s t i t u t e d

corresponding

3

1

regarding

a high

reactions

respectively

A S

t

been

at the

known

possessing

processes

0 H N ^ y V H ,

h n A

are

side-chain

in the a u t o x i d a t i o n

dialkyl-7 ,8-dihydropterins dehyde

also

stereochemistry

further

tic

ly b e e n

of u n k n o w n

( 1 6 ) , b u t no

the

have

6-(1 ' , 2 1 - d i h y d r o x y - 2

to t h e c o r r e s p o n d i n g

derivative

centres

side-chains

2 ,4-Diamino-6-styry1pteridine-8-oxide

H,N

XH,

•N^CH,

HN

Fig.

Results In o r d e r carbons simple and

to g a i n

reactions

benzyl

1960

that

the

information

position been

Henseke

to

treatment

the with

corresponding

can

7-bromomethyl

showing

and

Acid the

formation

about

7 of

out

and Muller

pyridine

nitrone.

phenylhydrazone

6 and

carried

1,7-dimethyl1umazine

ne-7-carboxaldehyde from

basic

at

have

groups.

in h i g h y i e l d quent

some

side-chains

have

methyl, already

brominated

derivative,

reactivity

pteridine

to m o d i f y (18)

be

the

the

ethyl, shown

in a c e t i c

which

on

normal (Fig.

led

carbonyl

6).

to

acid

subse-

p - n i t r o s o d i m e thy 1 a n i 1 i n e

hydrolysis

of

nucleus

formed

1-methyl 1umazi-

reactivity

as

seen

35

0

0

0

Fig. There

is a l s o

the

possibility

trimethyl1umazine and very

easily

oxaldehyde, inert

under

ly b a s i c

acid

medium,

the

of w h i c h

conditions

to

7-dibromomethyl

hydrolysis.

however,

gave

Reaction rise

tion with

could

formaldehyde

be g e n e r a t e d (Fig.

Fig.

7

directly

into

1,3,7the

6-

hydrolysed

1,3-dimethyl1umazine-6-carbfunction with the

by a c i d

7).

1,3,6- and

the 6 - i s o m e r

to t h e

thy11umazine-7-carbaldoxime , from which 7-carboxaldehyde

the

on b r o m i n a t i o n

derivatives,

acidic

whereas

towards

of c o n v e r t i n g

respectively

7-dibromomethyl

6

turned

o u t to

hydroxylamine

formation

of

in

be weak-

1,3-dime-

1,3-dime thy11umazinecatalysed

transoxima-

36 Analogously

the e x p e c t e d

1 ,3-dimethyllumazine hydrolysis. the

Whereas

dibromobenzoyl

l e d the

protic

higher

reactivity

was o b s e r v e d in

the case

derivative

work-up

of

of

could

of

the

reactivities

diamino-7-ethy1pteridine ferences drolysis whereas

in

showing

be i s o l a t e d

(Fig.

Fig. A comparison

also

always

latter

of

7 - (1 , 1 - d i b r o m o e t h y 1 ) - p t e r i d i n e

in c r y s t a l l i n e

to the

form

isolation

of

2 , 4 - d i a m i n o - 7 - b e n z y l - and

reveals

allows

7-benzyl-

subsequent

8

straightforward example

and

8).

bromination

the

as a s t a b l e

Fig.

9

in

dif-

and i m m e d i a t e

hy-

the former

isolation

2,4-

structural

the o b v i o u s

to 2 , 4 - d i a m i n o - 7 - b e n z o y l p t e r i d i n e the

t h e 6 - and

7-benzyl-1 ,3-dimethyl1umazine

the 6 - s i o m e r

6 - b e n z o y l - 1 , 3 - d i m e t h y l 1umazine

of

on b r o m i n a t i o n

of the

intermediate

case,

2,4-diamino(Fig.

9).

37 Direct

oxidations

different stituents. to

the

ah

alkyl

to

the

using

chemical The

benzyl

corresponding

oxy1ic

(Fig.

be

6- and

by

oxidative

the a - c a r b o n y l formation

of

illustrate and

nicely

derivatives

further

with

also

7-alkyl

oxidized

7-benzoyl

underlies

scission

permanganate

the

can

activation

in a C - C acid

of

groups 6- and

substituent

additional

finally

potassium

behaviour

in

high

sub-

yields

respectively , but t r a n s f o r m a t i o n due

function

the

the

aralkyl

resulting

corresponding

carb-

10).

CH,

KMnO T« j Levitt^ M • and Unden fr i end * S. J. Biol. 1964, 239, 2910.

Chem.,

3. Ichiyama, A., Nakamura, S., Nishizuka, Y. and Hayaishi, 0. J. Biol. Chem., 1970, 245, 1699. 4. Curtius, H.-Ch., Niederwieser, A., Viscontini, M., Otten, A., Schaub, J., Scheibenreiter, S. and Schmidt, H. Clin. Chim. Acta, 1979, 93, 251. 5. Narabayashi, H., Kondo, T., Nagatsu, T., Sugimoto, T. and Matsuura, S. Proc. Japan Acad., Ser.B, 1982, 58, 283. 6. Matsuura, S., Sugimoto, T., Murata, S., Sugawara, Y. and Iwasaki, H. J. Biochem., 1985, 98, 1341. 7. Matsuura, S., Murata, S. and Sugimoto, T. Chem. 735. 8. Matsuura, S., Murata, S. and Sugimoto, T. 1985, 23, 3115. 9. Bieri, J. H. and Viscontini, M. Helv. 1926 .

Lett.,

1984,

Heterocycles,

Chim. Acta,

1979, 60,

10. mp. 252-255 °C (dec); [a ] D 2 2 +49.63* (c = 0.524, 0.1 M HCl); pK, 1.25, 4.69, and 10.84; UV X «.ai/nm (log c ) at Hi -1.0: 262 (4.19); at pH 3.2: 264 (4.08), 300 (2.65); at pH 7.5: 260 (3.68),300 (9250); at pH 13.0: 289 (3.90);iH NMR (D 2 0) 1.25 (3H, d, J - 7.1 Hz, CHa ) , 3.60 (1H, dd, J - 14.7 and 9.0 Hz, C(7)H a x), 3.69 (1H, dd, J - 5.8 and 5.8 Hz, C(l')H), 3.73 (1H, dd, J - 14.7 and 3.4 Hz, C(7)H e q), 3.80 (1H, ddd, J - 9.0, 5.8, and 3.4 Hz, C(6)H), 3.94 (1H, dq, J - 5.8 and 7.1 Hz, C(2')H). Anal. Calcd for C 9 H i 5 N 5 0 3 • H 2 S 0 t : C, 31.86; H, 5.30; N, 20.64. Found: C, 31.62; H, 5.00; N, 20.34. 11. Oka, K., Kato, T., Sugimoto, T., Matsuura, S. and Nagatsu, T. Biochem. Biophys. Acta, 1981, 661. 12. Matsuura, S., Murata, S., Sugimoto, T., Sawada, M. and Nagatsu, T. Chem. Express, 1986, in press.

CHEMICAL SYNTHESIS AND PROPERTIES OF QUINONOID (6R)-DIHYDROBIOPTERIN

Sadao Matsuura, Shizuaki Murata, and Takashi Sugimoto Department of Chemistry, College of General Education, University, Chikusa-ku, Nagoya 464, Japan

Nagoya

Introduction Quinonoid (6R)-dihydrobiopterin catabolite

of

the enzymatic hydroxylation of living

cells,

has been assumed to

together

aromatic

amino

speculated

from

the

dimethyl analogue (1,2). thesis

and

(1).

This

In

the

recycling

ows the major responsibility

supply of the cofactor.

is such an important compound, only

acids.

with the reduction of 7,8-dihydrobiopterin by

dihydrofolate reductase, stationary

the

the catabolite (X) in turn becomes the substrate

of dihydropterin reductase to reproduce 2 system,

be

(6R)-5,6,7,8-tetrahydrobiopterin cofactor (2) in

properties

(6R)-dihydrobiopterin

the

but yet it has been

a

compound

studies on and the 6-methyl or 6,7We describe here

of

(JJ

for

Quinonoid dihydrobiopterin

hitherto and

the

the

chemical

syn-

uncharacterized quinonoid 6-methyl

and

6,7-dimethyl

analogues (7^,8).

Synthesis To

a

solution

of (6R)-tetrahydrobiopterin (2) dihydrochloride

(3) (1.26 g, 4.0 mmol) and 10% KI (64 n 1, ml),

30%

H202

( 400

ii 1,

4.0

mmol)

0.04 mmol) in HjO (10 was added at 0 • C under

stirring.

After 18 min the precipitate was collected by filtra-

tion

washed

and

with

H2O

and tetrahydrofuran to give 0.61 g

(55%) of quinonoid dihydrobiopterin (X) hydrochloride

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

as

ivory

82

needles (4)* By sin analogous wayf quinonoid 6-methyldihydropterin (7) hydrochlride (70%) and quinonoid 6,7-dimethyldihydropterin (8) hydrogen sulfate (10%) were obtained from the corresponding 5 t 6,7, 8-tetrahydropterins(5 ) . Chemical Properties The IR spectra (KBr) of the onium salts all exhibited a band at 1740-1750 cm - 1 which can be utilized as a diagnosis for the quinonoid dihydropterin structure. Although they decomposed quickly in solutions, the crystalline salts could be stored without significant decomposition at -10 *C for several months.

0 0 " S 0 Z OH

a

0

H

5

H

Scheme 1. The Transformation Paths of Quinonid (6R)-Dihydrobiopterin (¿). 0

0

7

H

8

H

83 The

quinonoid

(6J?)-dihydrobiopterin

(1_) followed several reac-

tion paths to give (6R)-tetrahydrobiopterin 7,8-dihydrobiopterin

(4),

in Scheme 1 and Table 1.

(2),

biopterin (3),

and/or 7,8-dihydropterin In a pH 6-7 phosphate

(5) as shown

buffer,

4

was

formed as the major product. The isomerization occured under almost

all the reaction conditions examined,

enhanced much by proton and phosphate ions basic

solution,

a

retro

3-4 ,

2 and

formed in equal decleased

on

In

a

more

aldol-type reaction to split off the

side chain predominatly occured giving pH

though the rate was (2a).

were the

(6).

major products.

quantities

and

their

Alternatively at Since 2

yields

and 3 were

relative

lowering the substrate concentration,

likely that a disproportionation of isomerization, produced 2 and

in

to

4

it is most

competition

to

the

3.

Table 1. Relative yields (X) of the decomposition products from quinonoid (6J?)-dihydrobiopterin (!).•> tetrahydrobiopterin(2)

pH 1.0

19

1 .0 •> ' 3.0C

7,8-dihydrobiopterin(3) biopterin(4) 22

6

7,8-dihydropterin(5)

44 9

none

85

none

41

45

10

none

6.0

8

10

66

16

11.0

3

4

18

75

HC1.

10 mM in 30 mM potassium phosphate buffer. c ' 100 mM in H 2 0 .

b

> 1 mM in 0.1M

All of these decompositions were well prevented by NaHS03, which

i formed a metastable adduct (6) across the C n - N s

with double

bond to exhibit a characteristic UV spectrum (7). Heating (5 with a large excess of NaHS03 yielded 2 and an unidentified (8) in nearly equal amounts.

compound

The adduct can be a model compound

for the primary adduct of the cofactor (2.) to O2 during the

en-

84 zyamtic hydroxylation. NADPH to

A nonenzymatic addition of hydrogen from

proceeded stereospecifically giving exclusively 2.

Quinonoid 6-methyl- (X) and analogous

isomerization

6,7-dimethyldihydropterin

into

(8)

the 7,8-dihydro isomers;

displayed a small extent disproportionation

into

did

T_ also

6-methylpterin

and its tetrahydro derivative in a pH 3-4 aqueous solution

(5).

References and Notes (1) a) Kaufman, S. J. Biol. S.J. Biol.

Chem.,

Chem.,

1964, 239,

1961, 236, 804; b) Kaufman,

332.

(2) a) Archer, M. C. and Scrimgeour, K. G. Can.

J.

Biochem.,

1970, 48, 278; b) Lazarus, R. A., DeBrosse, C.W., and Benkovic, S. J. J. Am.

Chem.

Soc.,

1982, 104, 6871.

(3) Matsuura, S., Murata, S. and Sugimoto, T. Chem.

Lett.,

1984, 26, 4003. (4) For the NMR spectrum, see: Matsuura, S., Murata, S. and Sugimoto, T.

Tetrahedron

Lett.,

1986, 27, 585.

(5) Matsuura, S., Sugimoto, T. and Murata, S. Tetrahedron

Lett.,

1985, 26, 4003. (6) iHNMR in D 2 0 : 7.03 ppm (1H, t, J = 3 Hz) and 4.17 (2H, d, J = 3 Hz). (7) A m a x at pH 3.0: 220 and 280 nm; HPLC retention time on a Partisil SCX-10

(4.5 x 250 mm) column eluted by 30 mM

ammonium phosphate, pH 3.0, containing 3 mM NaHSOs at 2.0 ml/min: 1.73 min. (8) X max at pH 3.0: 220 and 276 nm; HPLC under the same conditions as Ref.(7):

1.53 min.

PYRAZINE-RING CONFORMATIONS OF TETRAHYDROPTERINS AND QUINONOID-DIHYDROPTERINS

J. E. Gready Department of Biochemistry, University of Sydney, Sydney N.S.W. 2006, Australia

Introduction We have previously reported theoretical geometries at the SCF/STO-3G level for the neutral and ionised forms of tetrahydropterin and some quinonoid-dihydropterin tautomers

(1-3).

For the

tetrahydropterins these structures are in agreement with experimental x-ray

(4) and recent nmr (5) results in predicting a pyr-

azine-ring conformation distorted from a half-chair form such that only one ring atom

(C6) is significantly out-of-plane.

It

has been suggested that the flattened ring structure is due to the vinylogous-amide resonance which links the pyrazine and pyrimidine rings via the

C 4(04)C4aC8aN8(H8) group of atoms

However, as our related theoretical studies

(2,5).

(1-3,6) had indicated

that the presence and nature of the other ring in these pteridine derivatives influenced a number of structural and other chemical properties by complex

Ti-delocalisation

effects, we decided to

complement further investigation of this conformational behaviour with studies on tetrahydropyrazine and tetrahydropteridine.

In

this paper we have identified all possible pyrazine-ring conformers for H4pyrazine, H4pteridine and H4pterin at a higher theoretical level

(SCF/3-21G), and, for comparison purposes, those for

p-quinonoid-dihydropterin at the SCF/STO-3G level.

Methods and Results The conformer search and geometry calculations were performed with the GAUSSIAN-80 and GAUSSIAN-82 programs as reported previously (1,7).

As defined schematically in Figure 1 three stable half-

chair forms of H4pyrazine, two flattened conformers

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

(C6 out-of-

86

Figure 1. Structures for the three half-chair forms of H4pyrazine (a,b,c = H-C 1,2&3), and the two flattened conformers for H4pterin (d,e 5 1C6 S 2C6) and p-q-dihydropterin (f,g ï 1C7 &2C7). U, D and P denote whether the H5, C6, C7 and H8 atoms (or H4pyrazine equivalents) are up, down or within the general molecular plane.

Figure 2. ST0-3G geometries for the two conformers of p-q-dihydropterin shown in Figure 1. The angle sums around N8 are shown in brackets.

plane) for both H4pteridine and H4pterin, and two flattened conformers

(C7 out-of-plane) of p-q-dihydropterin were identified.

In Figures 2 and 3 the corresponding geometries are reported, including, in Figure 3, geometries for the tetrahydro forms in which the pyrazine-ring heavy-atom structure has been restricted to be planar.

These planar forms have been studied in order to

isolate "intrinsic" differences in ring-bond lengths, especially for C - N bonds, which we have previously shown

(7,8) depend on

the overall molecular topology and atom connectivity of these TT-W-bicyclic systems, from local conformational effects. In

87

Figure 3. 3-21G geometries for the planar (a), H-C 1,2,&3 (b,c,d) forms of H4pyrazine, and the planar and flattened conformers for H4pteridine (e,g,h) and H4pterin (f,i,j). The sum of the angles around the N5 and N8 atoms (and H4pyrazine equivalents) for the non-planar forms are shown in brackets.

T a b l e 1 the d e v i a t i o n s are s u m m a r i s e d .

(in 8) of the ring atoms

D e t a i l e d d i s c u s s i o n of the

from

planarity

conformational

88 a ,b

Table 1. Deviations(in 8) of Atoms from the Molecular Plane. H8 N8 H7 H7' H6 C7 H61 H5 C6 N5

0.35 1.43 0.03 -0.35 -1.43 -0.03 0.01 -0.36 0.07 -0.63 0.37 1.44 0.11 -0.37 -1.44 -0.11 -0.07 0.63 H-C 2 H-C 3 -0.04 0.30 0.24 1.30 -0.10 -0.51 -1.57 -0.29 -0.12 0.58

H4pyrazine H-C 1 -0.01 0.36

2C6

0.25 -0.22 -1.27 0.19 0.01 -0.01 0.03 -0.73 0.54 1.60 0.36 -0.14 -1.19 0.33 0.01 0.00

1C6

0.01 0.18 0.60

2C6

0.09 -0.74 0.70

H4pteridine 1C6

H4pterin

0.00 0.05 0.,49 1.56

p-q-H2pterin 1C7

0.04

2C7

0.07

1.67 0.43 -0.10 -1.14 0..35 0.03 0.07 1.75 0..62 0.03 -0.98 0..58 0.03 0.05

0..07 1.11 -0.,45 -0.57 -1.65 -0..36 0..05 -0.26 0..11 1.16 -0..40 -0.52 -1.60 -0..33 0..02 0.98

a Defined1 by C4a,C8a and the pyrimidine ring, or C1,C2 and the mid-point of H1&H2 for H4pyrazine. b Equivalent atom labelling for H4pyrazine as in Fig. 1.

energetics and H-H interactions will be given elsewhere:

here we

merely report that the SCF/3-21G energies for the non-planar conformers of H4pyrazine, H4pteridine and H4pterin are all approximately the same with the planar forms 7-9 kcal/mole less stable.

Discussion and Conclusions Focussing attention particularly on the C8a-N8 and C4a-N5 bond lengths as well as the angle sums around N8 and N5 as a measure of N-atom planarity we observe that: 1. The "intrinsic" tendency for a shorter bond length (la greater IT -electron délocalisation) for the C8a-N8 bond than for the C4a-N5 bond is present in the planar forms of both H4pteridine and (to an increased extent) H4pterin.

The C8a-N8 bonds, in

particular, are much shorter than for planar H4pyrazine. 2. For non-planar H4pteridine and H4pterin a possible four halfchair forms predicted on the basis of the H4pyrazine results have "collapsed" into only two forms in which the N8 environment is effectively planar and only the C6 heavy-atom is significantly out of the molecular plane.

This flattened ring

structure is thus not specifically due to the presence of the vinylogous-amide group in H4pterin.

N8 is more planar in these

89 3-21G geometries than in the previously reported

(1,3) ST0-3G

ones because of the improved representation of w-delocalisation effects by the larger basis set (6,7). 3. In agreement with earlier findings (1,3) the structures for the two non-planar conformers of p-q-dihydropterin are effectively non-aromatic, exhibiting little if any shortening of the C8a-N8 bond and no tendency towards planarity of N8.

The fact

that C7 is the heavy atom out-of-plane may be rationalised by assuming some residual TT -délocalisation preference for keeping N5 planar (and hence keeping the C4a-N5 double bond in plane) rather than N8. We conclude that the conformational complexity in these systems may be explained in terms of two competing energetic factors, H-H steric repulsions and stabilising délocalisation effects.

ir-electron inter-ring

The importance of this latter factor

m

imposing a flattened pyrazine-ring structure on H4pterin should apply also to most of the natural tetrahydrofolate cofactors.

Acknowledgement This research was supported by grants from NH&MRC and the Sydney Cancer Research Fund, and computing time from TAFENET(Sydney).

References 1. Gready, J. E.

1984. J.Mol.Struct.: THEOCHEM 109, 231.

2. Gready, J. E.

1985. J.Comput.Chem. 6, 377.

3. Gready, J. E.

1985. J.Am.Chem.Soc. 107, 6689.

4. Prewo, R., J. H. Bieri, S. N. Ganguly, M. Viscontini. Helv.Chim.Acta 6^5, 1094 and references therein. 5. Williams, T. C. and C. B. Storm. Int.J.Quant.Chem.

1982.

1985. Biochem. 24, 458.

6. Gready,

J. E.:

(in press)

7. Gready,

J. E. 1985.

J-Mol.Struct.: THEOCHEM 124, 1.

8. Gready,

J. E. 1984.

J.Comput.Chem. 5, 411.

HYDROGENATION OF SILYLATED PTERINS IN BENZENE SOLUTION

P.H. Boyle, M.J.Kelly University Chemical Laboratory, Trinity College, Dublin 2, Ireland

Introduction Reduction of the pyrazine ring of pteridines has been achieved using a variety of reducing agents, and the reaction is of great importance because it is in their reduced forms that naturally occurring pteridines such as folic acid or biopterin are biologically active. Because of the highly insoluble nature of most pteridines, catalytic reduction with hydrogen has usually had to be carried out in highly polar solvents such as aqueous acid or trifluoroacetic acid. This, however, has precluded the use of modern chiral hydrogénation catalysts, which are mostly used in organic solvents. We therefore investigated the hydrogénation of some model pteridine compounds in benzene solution by solubilising them as their trimethylsilyl derivatives. As well as achieving their reduction in organic solution, we also observed a novel de-silylation reaction of silylated pteridines, arising from their interaction in benzene solution with a soluble ligandcoordinated rhodium catalyst.

Results When a suspension of 6,7-dimethylpterin (_1) in benzene at room temperature was stirred in an atmosphere of hydrogen with a platinum catalyst, no absorption of hydrogen occurred. Neither was any reaction observed when a solution of its di-trimethylsilyl derivative (2) in dry benzene was stirred under hydrogen with the same catalyst. However, if benzene saturated with water was added slowly to this latter reaction mixture, absorption of hydrogen began immediately (see scheme 1), and a good yield of 6, 7-dimethy 1-5, 6 , 7, 8-tetrahydropterin (_3) separated

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

92 from

solution.

This

product was identical

with the

obtained by hydrogénation of 6,7-dimethylpterin oxide

in

spectrum groups

trifluoroacetic

acid solution

product

(1),over platinum,

(1),

and

its

p.m.r.

showed a cis disposition of the 6- and 7- methyl (2,3).

OSi (CH 3 ) 3

V r

N

Si (CH 3'3

Pt/H2

Pt/H2

HN H2N 1

9

CgHg+ H2O

H

/CH3 13)

N

H

N x

-

^ CH,

(Scheme 1

It

appears that

catalytic reduction of the pyrazine ring in

is inhibited by the adjacent bulky 4-trimethylsiloxy that

the

this

interfering group

derivative, would

group,

role of water in promoting hydrogénation is to by selective hydrolysis.

(2) and

remove

The monosilyl

6,7-dimethy1-2-trimethyIsilylamino-4-pteridinone,

thus be the species actually undergoing reduction.

Evidence

supporting

this idea was obtained

monosilylated compound, pteridinone

(i_) ,

by

preparing

the

3,6,7-trimethyl-2-trimethylsilylamino-4-

and hydrogenating it over a platinum catalyst.

Absorption of hydrogen occurred immediately without the necessity for the addition of any water

(equation 1).

93

C H

3 N ^ Y

N

Y

C H 3

R / H 2 i n

H N - ^ N ^ N ^ C H , I

C

C H

r

3 N ^ V N > Z h

6H6

3

K

Si (CH 3 )3 14) (Equation 1)

An

unusual

hydrogenate

reaction was discovered when an attempt was made

to

the

solution

in

No absorption

of

disilylated. pterin

(2^ in benzene

presence of the soluble catalyst Rh(DIOP)Cl. hydrogen

occurred.

conditions,

Instead,

6,7-dimethylpterin

even under rigorously

anhydrous

(jj was slowly precipitated from

solution in almost quantitative yield

(equation 2).

OSi (CH 3 ) 3

0

-CH3

R h l D l O P ) CI ^

H N ' ^ N ^

0

"

C6H6

12)

11) (Equation 2)

Control

experiments

occurred well

showed

that

in the complete absence

as

in

the

this

de-silylation

of water,

presence of hydrogen,

reaction

in the absence

of

Rh(DIOP)Cl.

The

reaction was markedly faster if thiophene was first added to

the

catalyst

catalyst solution. ution

with

silylation

contrary silylated

in benzene with thiophene alone,

free DIOP.

These results suggest that

is not caused by water hydrolysis,

the catalyst reaction),

with

De-silylation did not occur in benzene sol-

without catalyst,

benzene

as well as

the

as

use

[Rh (COD)DIOP] + C10 4 ~

and with

(since

the

the de-

by chloride

perchlorate catalyst

or by free DIOP in the catalyst.

also

gives

and

the catalyst,

from the

They suggest on the

that the reaction is caused by interaction between pterin

or in

and furthermore,

the

that the

94 catalyst

is

most

thiophene). transfer

A

active mechanism

in its is

monomeric

proposed

in

form

(effect

scheme

of an "unactivated" hydrogen atom from a

2

silyl

methyl

group to the rhodium atom of the soluble catalyst.

H-CH2

cr R/h ^ g V S i ( C H )

3 2

S

n^VnYCH3 3

HNVr

CH3 +

DIOP \ / Rh

H N ^ N ^ N ^ C H 1

Si (CH 3 ) 3

S = Solvent

/ \

S

CI

Scheme 2

ABBREVIATIONS DIOP

=

2,3-0-isopropylidene-2,3-dihydroxy-1,4-bis(diphenylphosphino)butane

COD

=

1,5-cyclooctadiene

REFERENCES 1.

Viscontini, M. and J. Bieri. 1972. Helv.Chim.Acta

2.

Armarego, W.L.F. and H. Schou. 1977. J. Chem. Soc., Perkin Trans. 1, 2529

3.

Weber, R. and M. Viscontini. 1976. Helv.Chim.Acta

of

showing

55,21

59,2379

N(5)-ALKYLATION OF POLYACEIYLATED 5,6,7,8-TETRAHYDROPTERINS

S. Antoulas Zentrale Forschungseinheiten, F. Hoffmann-La Roche Inc. CH - 4002 Basle M. Viscontini Organisch Chemisches Institut, Universitaet Zuerich-Irchel CH - 8057 Zuerich

Introduction The diastereomeric mixture of 6 a - and 6 P -tetraacetyl-tetrahydro-D-neopterin (D-l)^

gave

under

hexaacetylated

drastic

derivatives

acetylation

a mixture

of

conditions 6a-

besides

N,2-N,8-heptaacetyl-5-ethyl-5,6,7,8-tetrahydro-D-neopterin 0

H

A JL

OAcOAc -CH 2 0Ac

"

*=20' p>,ridin

penta-

and

and 6 p -1'-0,2 1 -0,3'-0,4-0,2(D-2) (Scheme X).

OAc ^ OAcOAc ^ N y f - C -CH 2 0AC

1 1/2 h reflux

A A

Ac-N^N t^VH ! ' H Ac Ac 6(a+p)-D-2

HN-^-N-^N^TH 1 u H Ac H 6{a + p)-D-1

"

Scheme 1 The 1

structure

of

the (3 -diastereoisomer

6 p-D-2 has been established by

H-NMR, MS and X-ray diffraction analysis [1].

Mechanistic Considerations Ethanol, which was used during work up, can be excluded as a possible source of alkylation. In a control experiment using C^J^CD no deuterium incorporation was observed.

The terms and are used according to IUPAC rules for the nomenclature of steroids as proposed by M. Viscontini [2].

Chemistry and Biology of Reridines 1986 © 1986 VVaiter de Gruyter & Co., Berlin • New York - Printed in Germany

96 A careful examination of the reaction mixture (before and after work up) by 2) TLC revealed, that oxidised products were formed as well As a result of these observations, two reaction mechanisms, based on the property of reduced pterins to act as hydride donors [3], are proposed. Proposed rrechanism 1

H

e-

TIT ¥ N^R

1nr

^

y

0—C-OI® I 1

XiT

- H

Ac

Ac

Ac

•2 H®

-H20

CH,

Ox. Pterin H®

E»

Red. Pterin

0 Ç* nÀ^NV^-R ( AC>7 N

(Ac)2N^N

N

CHjCO

iH3 AcO *CH

H H

(AcljN

jnrr

Red. Pterin

Ox. H® Pterin

Ac

^

Scheme 2 Intermolecular redox reaction with participation of the amide function of the pyrimidine ring

2)

All reactions were carried out under nitrogen, oxidation by air can therefore be excluded.

97 Proposed mechanism 2

• H® - CH3COOH

CH,

CH3

H-FVoAC

IT

®Ç VOAc -N^-^OAc

OAc H

Ox. Pterin Red. Pterin

J

-N' Ac

CH 3 CO®

ÇH3

tr H-C*

Red. Pterin

-Ox. hPPtecki

C,H, 2n5

X»T 1

Ac

Ac

6 ( a •*• P l - D - 2

Scheme 3 Intermolecular redox reaction with participation of the side chain of the octaacetyl-tetrahydro-D-neopterin

The intramolecular ringformation depicted in Scheme 3 is arbitrarily shewn with participation of the acetyl group at C(3'). A participation of the acetyl groups at C(2*) or C(l') cannot be excluded.

98 Control Experiments and Conclusions Acetyl-tetrahydropterin, which does not possess a side chain, undergoes no alkylation under the same acetylation conditions. Therefore irechanism 1 would appear unlikely for the ethylation of D-l. Preliminary experiments dealing with the behaviour of (6 a + P )-triacetyltetrahydro-L-biopterin under drastic acetylation conditions indicate, according to masspectroscopic analyses, that a 5-ethyl-hexaacetyl-derivate of tetrahydro-L-biopterin is formed. These findings are compatible with a mechanism 2 like reaction, in which acetyl group C(2') or C(l') is participating in the manner indicated in Scheme 3. Further experiments are necessary for a definite elucidation of this reaction mechanism.

Acknowledgement s The authors wish to thank Prof. Dr. H.J. Hansen and Drs. M. Schmid and R. Schmid for helpful discussions, Drs. G. Englert and W. Arnold for the NMR spectra, Dr. W. Vetter and Mr. W. Meister for MS measurements and F. Hoffmann-La Roche Inc. (Basle) for financial support.

References [1] S. Antoulas, R. Prewo, J. Bieri & M. Viscontini, Helv. Chim. Acta 69, 210 (1986) [2] S.N. Ganguly & M. Viscontini, Helv. Chim. Acta 67, 166 (1984) M. Viscontini. 1985. In: Biological and Clinical Aspects of Pteridines (W. Wachter, H.Ch. Curtius, W. Pfleiderer, eds.). Walter de Gruyter & Co., Berlin, New York, p. 57 [3] F. Stierli, J. Bieri & M. Viscontini, Chimia 38, 429 (1984)

OXIDATIONS PHOTOSENSITIZED BY PTERINS AND DIAMINOPTERIDINES

M. Aubailly, R. Santus Laboratoire de Physico-Chimie de l'Adaptation Biologique U.A. CNRS n° 481, Muséum National d'Histoire Naturelle 43, rue Cuvier, 75231, Paris Cédex, France.

Introduct ion In

some

animals, pterins are concentrated in areas

light (skin, tegument) or in gland).

This

to

led us to investigate the photochemical properties

of these derivatives. diated in

exposed

light sensitive organs (eye, pineal

Some time ago,

we showed that, when irra-

its first absorption band,

2-amino-4-pteridinone

sensitize the oxidation of amino acids and purine bases,

can

via the

usual type I and type II mechanisms (1). The aim of this paper is to

present both new data and a comparative study of pterins

their analogs, the 2,4-diaminopteridine derivatives

and

(R-DAP).

Results 1)

oxidations by the triplet excited state (Type I photosensiti-

zat ion ) . According to this mechanism, between

there is a direct electron transfer

the substrate and the sensitizer in its triplet

excited

state

(2). In a first time, the characteristics of the triplet 3 * state ( P ) must be studied. This has been done using laser flash spectroscopy (the experimental set-up and method for the determi-

nation

of

described DAP),

a

the triplet state characteristics have ( 1 )J .

already

2,4-Diamino-6-pteridinecarboxaldehyde

(6-CH0-

methotrexate photoproduct (3) was chosen as a model

2,4-diaminopteridine

derivative.

Fig.l

shows

the

absorption

spectrum of the triplet state observed 0.2

the

flash.

laser

substrate

been

cation

laser flash.

The

pteridine

radical MeTRP'

The lifetime of the

+

anion are

after the

observed 16 ys after

the

state is

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

P"

ys and

triplet

radical

of

transient

reduced

as

100

Fig. 1 Transient

absorption

measured 16

0.2 ps (

spectra

— ) and-

ys(-o- ) after the laser

flash ( 353 nm excitation)

0.10 .

(6-CHO-DAP) = 1.5 10

M

(1-Me-Tryptophan) = 5 10 pH = 7 (10~

2

M phosphate

argon saturated

0l05

400

500 M nm )

M buffer)

solution.

600

compared with the absence of oxidizable substrate. This data provides

evidence for the type I

experiments

have

characteristics

photosensitized

oxidation.

enabled us to obtain results of Table of

the pterin triplet state (1) are

Such

1.

The

given

for

comparison. Table 1 : Triplet State Characteristics of 6-CHO-DAP and Pterin T 1.5

= triplet state lifetime (ground state concentration = -4 x 10 M), $ = triplet state production quantum yield.

= X

6-CHO-DAP

Max

(nm)

:

e

Max

(M

X

cm

1

)

: T t (ys)

:

415

:

11 000

:

3.4

: 0 60

pterin

:

550

:

2 000

:

2.3

: 0 23

(pH = 9.2)

:

350

:

4 700

(pH = 7)

101 2)

Oxidations involving singlet oxygen (Type II

photosensitiza-

tion ) . In

this mechanism the oxidant is singlet oxygen "^C^ .

duced

by

energy transfer between ground state

It is pro-

oxygen

and

the

sensitizer in its triplet excited state (2). Histidine is readily oxidized shown

by

^0-,- Using this amino acid as a 1

that

substrate

pterins and 2,4-diaminopteridines can give

they are U.V.

irradiated.

we have

1

This is a common feature of

when unconju-

gated pteridines only (Table.2). Methotrexate and triamterene are not photosensitizers. Table

2.

Photooxidation

Presence

of

Pterins

Sensitizers. saturated

or

Irradiation

solutions,

Quantum

Yield

of

Histidine

2,4-Diaminopteridines wavelength 10

PH

2

\ = 366 nm,

M

phosphate

in

the

(R-DAP)

as

oxygen buffer

(Histidine) = 5 10~ 4 M. 6-CHO

6-COOH

6-CH 2 OH 6,7di-i-Pr methotrexate:triamterene

DAP

DAP

DAP

DAP

0 .050

0.035

0.019

0 .035

1 OI O S-

O

V)

o >-.| í/) 00

o m

cu > o>


M UJ CD O — O i t < 1— oo Lü < I— _i < o. O Lü z Z o 1 O h- z Lü c OC 1 O X «t LÜ s : a: * o 00 z hz: •z. t « Lü — hz o C¿

«3 u 23 o

Oc CO (O O) •r- ^ S- +J +J c O l-H u CL E a. 00 a> c o co c •o a ra cu q- s- c o 4-> E m o J- X O ai

CSJ

IT)

- L U X hO O. a: JJ • f a >- -w >-1 O l X HE"-» » • M ( O O l « O S -M vo Z a » e t o

•» +

L T ) O O

o i r > ro

+

« s vo *—

o r » +

w

+

i / ) O) t. +

c

< / l a > L.

ut o > l O

•

00 C M

•w

O ID 00 C M V —

o C M

m r-.

L T > L f ) m

o

CD

o a>

C M O C M

—

C

C M

•

O z HC M «— Zz < —" X cc OL U l / > h> •C L . Z >—« 1 Û C OL k J hco a. z 1 M

» 4) un U-—• o O« C i / i z o> c a. a > oi — t. 1 Û£ OL U E ' O 3 L U hz a. « O i— • M C < 9 t - ¿Z a i -m 1 / 1 o » a > i o X c • r -< —o T3 O J • < - .o a. i - i - i a > o i lA •m . e a. r r— o. a. oc L O 0) 4 » o ( / I r a »— o -O o N. c e n o O. z

o r > » o

O Oí

o < x > < N J C M

o C M

1 / 1

a J Î.

L f > c r .

c

en

C M

< T >

en

•

o 00

o\

,—

V I 0)

r s . C M

+

c o m o m


• M < / > «/» T J f T J < O — C» — p W. O . Û O O o Cl c c 3 o o < / > s Z

o

O r-.

00 i T )

« o E E < T J o> z l i .

m E E r o a» z u. « 4 -« O + • M C

en ifí

Q. o • f + J c v i 4 > < / > c« — u .O 0) o Q. C 3 O oo E

>—
^ ' — N E • — >• — 3 Z" O Q- " O Q£ X a > O E » E a » r3 C E • r -i —\ " O" O en 0) O Q. Z —'

438 fluoresceins by V i s c o n t i n i et a l . (_1£,

). In Comamonas s p . , L-monapterin was r e -

ported to be a cofactor involved in the oxygen-dependent hydroxylation of phenylalanine (J_2). Although Gene M. Brown reported an enzyme-dependent epimerization of dihydroneopterin triphosphate to dihydromonapterin triphosphate in E. c o l i ("D-erythro-dihydroneopterin triphosphate 2 ' - e p i m e r a s e " ) , the biochemical

func-

tion of L-monapterin in E. c o l i remained unknown (J_3). D-erythro-Neopterin, L-monapterin and L-monapterin 2 ' 3 ' - c y c l i c phosphate were i d e n t i f i e d in S e r r a t i a indica as products from GTP; however, an pteridine triphosphate from the enzymatic reaction of i s o l a t e d S. indica GTP-CH, which was also found, was reported to be different from D-erythro-dihydroneopterin triphosphate ( U K

and the question

a r i s e s as to whether i t could be L-dihydromonapterin triphosphate. The reduced form of L-monapterin was only found to be active as a cofactor of phenylalanine hydroxylase in rat l i v e r (14). In human or animal c e l l s , however, we know almost nothing about the biosynthesis and the biochemical role of the L-threo-neopterin

(L-monapterin).

In our i n v e s t i g a t i o n s on human blood c e l l s and body f l u i d s , we have demonstrated the simultaneous occurence of neopterin and monapterin as well as t h e i r nearly constant r a t i o in the p a r t i c u l a r compartment investigated (see Table 2 ) . Analogous f i n d i n g s were obtained in patients s u f f e r i n g from malignant diseases (tumor, leukemias): a l t e r a t i o n s in neopterin were accompanied in most cases by analogous a l t e r a t i o n s in the monapterin l e v e l s before and during treatment with c y t o s t a t i c s and/or a n t i b i o t i c s

(15-18).

Proglicem, which affects the b e t a - c e l l s in the human pancreas and induces hyperglykemia, caused a s i g n i f i c a n t decrease in both pterins in the peripheral

leuko-

cyte f r a c t i o n , the plasma and the urine (19). The c h a r a c t e r i s t i c l e v e l s and r a t i o s of neopterin and monapterin in human c e l l s and body f l u i d s ( i n healthy adults) are summarized in Table 2. The important f i n d i n g s are as f o l l o w s : - Neopterin and monapterin seem to have nearly constant r a t i o s in a l l

compart-

ments ; - Neopterin was found in higher concentrations than monapterin in a l l the cases investigated; - The lowest concentration of monapterin in r e l a t i o n to neopterin was observed in the urine (N/M, approx. 10). In other body f l u i d s (plasma, CSF) and in the peripheral erythrocytes, the r a t i o (N/M) was about 6. In c o n t r a s t , c e l l s which probably have a marked pterin metabolism (eg. bone marrow, peripheral

lympho-

cytes and granulocytes) demonstrated r e l a t i v e l y high concentrations of monapterin (N/M, approximately 3).

439

Table Z .

PTERIDINES

IN HUMAN CELLS AND BODY

FLUIDS.

R p - H P L C a n a l y s i s of i o d i n e - o x i d i z e d (pH = t ) and d e p r o t e inized s a m p l e s (fluorescence d e t e c t i o n , e x c / e m = 3 6 0 / 4 6 0 nm). For details see "Methods and M a t e r i a l s " . NEOPTERIN (TOTAL)

MONAPTERIN (TOTAL)

RATIO V«I

ERYTHROCYTES, peripheral b l o o d (ng/ml e r y t h r o c y t e s )

2.4

0.4

6.0

RED C E L L S , bone m a r r o w (ng/ml red cells)

4.6

1.1

4.2

WHITE CELLS, peripheral blood (Lymphocytes, 51%, Monocytes, 51, Granulocytes, 40«, Others, 4%)

296

48

6.0

«

WHITE C E L L S , bone m a r r o w

620

154

4.0

LYMPHOCYTES, peripheral blood

o

1228

393

3.0

THROMBOCYTES, peripheral blood

E

37

8

4.6

315

92

3.4

>.

a

GRANULOCYTES, peripheral blood PLASMA (ng/ml

plasma)

2.8

0.4

7.0

C E R E B R O S P I N A L FLUID (pg/mg l y o p h i l i z e d CSF) *'

543

93

6.0

URINE (nmol/mmol

311

32

10.0

creatinine)

•) ' 1 mg l y o p h i l i z e d

A C S F — 100 mg cerebrospinal

fluid

(n*27).

In freshly prepared peripheral cell fractions (erythrocytes, white cell fraction) obtained from two blood donors, reduced forms of neopterin and monapterin were not detectable. The highest concentrations of reduced neopterin and monapterin were observed in the CSF of healthy individuals (see Table 3). Table

3.

REDUCED

PTERIDINES

IN

BIOLOGICAL

MATERIALS.

The v a l u e s a r e the d i f f e r e n c e s b e t w e e n the r p - H P L C a n a l y s i s of a c i d i o d i n e - o x i d i z e d s a m p l e s (total a m o u n t of p t e r i d i n e s ) a n d of n o n - o x i d i z e d s a m p l e s ( o x i d i z e d p o r t i o n of p t e r i d i n e s ) . For d e t a i l s see " M e t h o d s a n d M a t e r i a l s " . NEOPTERIN (REDUCED)

MONAPTERIN (REDUCED) %

ERYTHROCYTES , peripheral blood WHITE CELLS, peri phera1 blood PLASMA CEREBROSPINAL URINE

FLUID

...

...

...

54

54

88

86

57

70

440 Our r e s u l t s lead us to the following questions: i ) I s there a "D-erythro-dihydroneopterin triphosphate 2'-epimerase" in human c e l l s , as we suggested some years ago (V7> J8.) > and/or i i ) I s there a further GTP cyclohydrolase, the a c t i v i t y of which leads to phosphorylated dihydromonapterin (triphosphate ? , 2 ' 3 ' - c y c l i c phosphate ?) ?

Acknowledgement We are indebted to the Deutsche Forschungsgemeinschaft for f i n a n c i a l

support.

References 1. Z e i t l e r , H . - J . , B. Andondonskaja-Renz. 1986. In: Methods in Enzymology (S.P. Colowick, N.O. Kaplan, F. Chytil and D.B. McCormick, e d s . ) . Academic Press, Orlando, p. 273. 2. Z e i t l e r , H . - J . , B. Andondonskaja-Renz, G. KUther, A. Struppler. 1986. I n : Chemistry and Biology of P t e r i d i n e s ; Pteridines and F o l i c Acid Derivatives (B.A. Cooper and M. Whitehead, e d s . ) . De Gruyter, Berlin-New York, in press. 3. Huber, Ch., J.R. Batchelor, D. Fuchs, A. Hausen, A. Lang, D. Niederwieser, G. Reibnegger, P. Swetly, J. Troppmair, H. Wächter. 1984. J. Exp. Med. J 6 0 , 310. 4. Takigawa, S . , H.-Ch. C u r t i u s , U. Redweik, S. G h i s l a . 1986. Biochem. Biophys. Res. Commun. 134, 646. 5. Sh intaku, H., H.-Ch. C u r t i u s , A. Niederwieser. 1986. 5th International Workshop on Biochemical and C l i n i c a l Aspects of P t e r i d i n e s . S t . Christoph, Arlberg, A u s t r i a . 6. Fuchs, D., A. Hausen, H. Lutz, G. Reibnegger, E.R. Werner, H. Wächter. 1985. In: Biochemical and C l i n i c a l Aspects of Pteridines (H. Wächter, H.-Ch. Curt i u s and W. P f l e i d e r e r , e d s . ) . De Gruyter, Berlin-New York, p. 287. 7. Stea, B., R.M. Halpern, B.C. Halpern, R.A. Smith. 1981. C l i n . Chim. Acta JM3, 231. 8. Wächter, H., A. Hausen, K. Grassmayr. 1979. Hoppe-Seyler's Z. Phys. Chem. 360, 1957. 9. Dhondt, J . L . , J.M. Hayte, C. Noel. 1985. In: Biochemical and C l i n i c a l Aspects of Pteridines (H. Wächter, H.-Ch. Curtius and W. P f l e i d e r e r , e d s . ) . De Gruyter, Berlin-New York, p. 419. 10. V i s c o n t i n i , M., M. Pouteau-Thouvenot, R. BUhler-Moor, M. Schroeder. 1964. Helv. Chim. Acta 47, 1948. 11. V i s c o n t i n i , M., R. Provenzale. 1968. Helv. Chim. Acta 5J_, 1495. 12. Guroff, G., C.A. Strenkowski. 1966. J. B i o l . Chem. 241_, 2220.

441 13. Brown, G.M., J. Yim, Y. Suzuki, M.C. Heine, F. Foor. 1975. In: Chemistry and Biology of Pteridines (W. P f l e i d e r e r , ed.). De Gruyter, Berlin-New York, p. 219. 14. Iwai, K., M. Kobashi. 1975. I n : Chemistry and Biology of Pteridines (W. P f l e i d e r e r , e d . ) . De Gruyter, Berlin-New York, p. 341. 15. Andondonskaja-Renz, B . , H.-J. Z e i t l e r . 1984. I n : Biochemical and C l i n i c a l Aspects of Pteridines (W. P f l e i d e r e r , H. Wächter and H.-Ch. C u r t i u s , e d s . ) . De Gruyter, Berlin-New York, p. 295. 16. Andondonskaja-Renz, B . , H.-J. Z e i t l e r . 1985. In: Biochemical and C l i n i c a l Aspects of Pteridines (H. Wächter, H.-Ch. Curtius and W. P f l e i d e r e r , e d s . ) . De Gruyter, Berlin-New York, p. 559. 17. Z e i t l e r , H . - J . , B. Andondonskaja-Renz. 1984. In: Biochemical and C l i n i c a l Aspects of Pteridines (W. P f l e i d e r e r , H. Wächter and H.-Ch. C u r t i u s , e d s . ) . De Gruyter, Berlin-New York, p. 313. 18. Z e i t l e r , H . - J . , B. Andondonskaja-Renz. 1986. In: Cancer Detection and Prevention (The International Society for Preventive Oncology) (H.E. Nieburgs, e d . ) . A.R. L i s s , New York ( i n p r e s s ) . 19. Z e i t l e r , H . - J . , B. Andondonskaja-Renz. 1985. Unpublished data. 20. Oyer, P.E., S.W. Jamieson, E.B. Stinson. 1982. Heart t r a n s p l a n t . U

285.

SUMMARY PTERINS AND HUMAN ILLNESS

D. Fuchs, H. Wachter Institute for Medical Chemistry and Biochemistry University of A-6020 Innsbruck, Austria One of the major recent advances in the field of pterin gation

has

been

the

investi-

recognition that pterins are produced and

excreted during immune responses particularly associated with the activation of the cell-mediated The

observation

immunity.

that this activation of the cellular immune sy-

stem is paralleled strictly by e.g. elevated production pterin

both

in

of

neo-

vitro and in vivo has a number of useful impli-

cations in clinical chemistry. The physiological conditions

lea-

ding to neopterin production have been partly explored. Activated T-lymphocytes secrete interferon-gamma that to

induces

macrophages

produce neopterin by GTP cyclohydrolase via dihydroneopterin-

triphosphate. Whether this reduced or oxidized neopterin plays role

a

in cell-mediated immunity still remains a matter of contro-

versy and speculation. The studies were performed mainly by HPLC with tection

based

on

fluorescence

out preceding oxidative step. This procedure does dihydro-

and

de-

direct measurement of urinary neopterin with-

tetrahydroneopterin.

not

determine

Similarly the application of

radioimmunoassay for neopterin in serum is usally conducted without

oxidation

steps.

However,

techniques

involving oxidation

steps of dihydro- and tetrahydroneopterin appear to produce

com-

parable clinical results to those without such steps. In

this chapter, the diagnostic potential of neopterin assays is

illuminated on a few presented more,

the

examples

of

diseases.

Further-

dependence of reduced and oxidized forms of neopterin

in healthy subjects and in patients with diseases involving activation

of

the

cellular immune system are studied. In this con-

text, the concomitantly measured activity of and

GTP

cyclohydrolase

intracellular levels of GTP in macrophages and T-lymphocytes

appear to be of interest as this study possibly

contributes

in-

formation on the physiological role of neopterin production. Two

further reports focus on some of the most prominent clinical

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

444 applications of the neopterin assay, the monitoring of the clinical

status

of patients having received a bone marrow transplant

and the condition of individuals infected with human

immunodefi-

ciency virus. Finally,

the

content and excretion patterns of other pteridines

besides neopterin in immune competent cells was reversed

investigated

by

phase HPLC subsequent to iodine oxidation with particu-

lar emphasis to micro techniques.

SECTION D FOLATES AND PTERINS IN MAMMALIAN TISSUES

FOLATES IN TISSUES AND CELLS. SUPPORT FOR A "TWO-TIER" HYPOTHESIS OF REGULATION OF ONE-CARBON METABOLISM.

Carlos L. Krumdieck and Isao Eto Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, AL 35294

Introduction In 1946 Pfiffner et al (1) and Angier et al (2) published in Science the first reports demonstrating the existence of polyglutamyl peptide derivatives of folic acid.

Two years later, the

exact configuration of the peptide chain was determined by comparison to synthetic isomeric peptides (3).

The year of 1986

marks, therefore, the fortieth anniversary of the discovery of the poly-y-glutamates of folic acid.

I will take this occasion to

focus my remarks on these, the true naturally occurring folates in tissues and cells. For many years, we (4-6) and others (7-10) have investigated the hypothesis that changes in the length of the polyglutamyl chain serve as an element of regulation of one-carbon metabolism.

The

strongest evidence supporting this view comes from in vitro work by a number of investigators demonstrating that the kinetic parameters for folyl polyglutamate substrates and inhibitors (516), and even the mechanisms of catalysis of folate-requiring enzymes (8) are, in most cases, dramatically altered by changes in the length of the polyglutamyl chain.

In addition, it has been

shown that pteroyl-polyglutamates are preferentially "channeled" through two multifunctional proteins of folate metabolism (17,18) and possibly also between enzymes involved in de novo purine biosynthesis (19).

Since channeling is thought to be kinetically

advantageous, the one-carbon flux through these reactions could be modulated by altering the chain length of the polyglutamates.

A

mechanism for the regulation of the activity of folate-requiring enzymes based on covalent modification of their cofactors (and/or inhibitors) by addition or deletion of Y-glutamyl residues (5) seems, therefore, a likely possibility.

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

448 The in vitro demonstration that the catalytic efficiency of folate-dependent enzymes is affected by the chain length of the cofactors is a necessary, but insufficient, condition to validate the regulation hypothesis.

It is necessary also to demonstrate

that changes in the chain length distribution of the folates occur in vivo in response to alterations in the steady state of onecarbon metabolism.

Considerable difficulty has been encountered

to demonstrate this, primarily because of limitations of the analytical methods.

I will briefly review some recent advances in

methodology, and new evidence supporting the regulation hypothesis, and offer in the end a possible explanation for why the folate-requiring enzyme systems, which respond to classical regulatory mechanisms, would have to be subjected to yet another, seemingly redundant, regulatory system.

Analytical methods No single method has yet been devised that is applicable to the determination of all known naturally occurring pteroyl mono and polyglutamates (20,21).

Some remarkably successful efforts

combining ion exchange chromatography, gel permeation and differential microbiological assays before and after conjugase treatment, resulted in the quantitation and chain length determination of many of the one-carbon substituted and unsubstituted, reduced and oxidized, folates of a limited number of biological samples (20).

These methods are however so laborious and time consuming

and so subject to the drawbacks of microbiological assays, that they have been essentially abandoned

in favor of simpler, though

often less informative, procedures. Most, if not all, modern methodologies resort to analytical approaches that limit the complexity of the family of natural folates.

The most often used approach has been to cleave the C 9 -

N^g bond converting the folyl polyglutamates (PteGlu n ) to a far simpler homologous series of p-aminobenzoyl polyglutamates (pABGlu n ) easily resolved and identified by co-chromatography with authentic synthetic markers.

The drawbacks of earlier oxidative

and reductive cleavage procedures (20), which failed to cleave several of the naturally occurring folates, partially degraded the

449

pABGlu n , and introduced errors in quantitation due to the presence of uncleaved [31,5',7,9(n)-^H] folates contaminating fractions presumed to contain only 3',5' labeled pABGlu n , were recognized by Maruyama et al (22) and by Lewis and Rowe (23). Newer cleavage procedures (24-28) have resolved the above difficulties by combining oxidative and reductive treatments and by increasing the sensitivity of the pABGlun quantitation methods by forming azo dyes (24) or by post-column fluorescamine derivation (27).

This eliminated the need for radiolabeling the folates of

the sample and permitted the analysis of "endogenous" patterns. Although much valuable knowledge has been gained by the application of Cg-N^o cleavage procedures, it is obvious that this approach sacrifices all information relative to the one-carbon substituents.

In an effort to recover some of this information,

we have taken advantage of the different susceptibility to oxidative cleavage of the various reduced, one-carbon substituted and unsubstituted folates to develop a "differential cleavage" procedure that permits the quantitation and determination of chain length of three pools of the folates (29-32).

Pool 1 contains

dihydro and tetrahydro pteroyl polyglutamates

(H2PteGlun,

H 4 PteGlu n ) plus 5,10-methylene-tetrahydro folates (5,IO-CH2-H4PteGlun); pool 2 is made up solely of 5-methyl-tetrahydro folates (S-CHj-H^-PteGlUjj), and pool 3 includes all reduced folates with substituents at the formyl oxidation level: 5,10-methenyl- (5,10CH=H 4 -PteGlu n ), 5 and 10 formyl- (5 and 10-CHO-H4-PteGlun) and 5formimino-tetrahydro folates (5-CH=NH-H4~PteGlun).

Conditions

that selectively cleave the folates of pool 1, of pools 1 + 2 , and of all three pools 1 + 2 + 3 ,

have been found.

The pABGlu n , after

formation of their azo-dye derivatives (Azo-pABGlun) by the Bratton-Marshall procedure (24) are pre-purified on a polyacrylamide column, concentrated by the use of a disposable octadecylsilane column and separated by reversed phase HPLC (33).

An

important refinement of this methodology has been the synthesis of two internal standards, Azo-p-aminobenzoyl-a-glutamyl-Y-glutamylglutamic acid and Azo-p-aminobenzoyl-a-glutamylglutamic acid, which have the same molar extinction coefficient as the AzopABGlun derived from natural folylpolyglutamates, and are readily separated from them by the HPLC column.

Inclusion of these

450 compounds improves the quantitation and facilitates the identification of sample-derived Azo-pABGlun (33). The most promising new methodology is that introduced by Priest et al (34,35) in 1980. Their method is based on the formation of a ternary complex between 5,10-CH2"H4-PteGlun, thymidylate synthase (T.S.) and radiolabeled 5-fluoro-2-deoxy[3H]uridylate ([3H]FdUMP). The use of the enzyme T.S. as a reagent which very selectively and efficiently extracts and incorporates the 5,10-CH2"H4PteGlun of a sample into a radioactively labeled, high molecular weight, covalently linked stable complex, is central to the success of this method. Its sensitivity is extremely high and limited only by the specific radioactivity of the [ 3 H]FdUMP used. The negative charge of the ternary complex increases by one with each successive ^-glutamyl residue. This gives the complexes of the various polyglutamates different electrophoretic mobilities which are a linear function of the number of glutamyl residues. If the experimenter is not interested in determining the length of the polyglutamyl chain, the complexes can be purified free of excess [ 3 H]FdUMP by gel permeation and the concentration of 5,10-CH2"H4PteGlun be determined by radioactivity counting. To quantitate and determine the chain length of other 1-C substituted and unsubstituted pools, Priest and his collaborators have developed enzymatic procedures that convert specific folate pools to 5,10-CH2"H4-PteGlun. Figure 1 summarizes the principles employed. In most cases a complexation step with non-radioactive FdUMP, designed to remove the endogenous pool of 5,10-CH2"H4PteGlun, is inserted prior to conversion of the pool to be quantitated into the T.S.-reactive 5,10-CH2"H4PteGlun. The direct estimation of H4PteGlun is obtained by the inclusion of formaldehyde (35) as shown in Figure 1. Adding formaldehyde to the nonradioactive complexation mixture removes both endogenous 5,10-CH2~ H4"PteGlun and H4"PteGlun, setting the stage for the determination of 5-CH3-H4"Pte-Glun after its methylene reductase-mediated oxidation to 5,10-CH2"H4-PteGlun in the presence of menadione (36). Dihydrofolates and fully oxidized PteGlun can be independently determined by converting them to H4"PteGlun with dihydrofolic reductase. At low reductase levels H2~PteGlun, the preferred substrate, is quantitatively converted to H4~PteGlun with little

451

Ternary Complex Assay: Principle * FdUMP T.S. Non-radioactive complexation removes: X=H 4 X=5,10-CH2 - H < X=5,10-CH 2 - H 4 *

Tissue folates: X

H C H 0

[ 3 H] FdUMP

V/

>/

y/

s/

V/

N/

Thymidylate Synthase (T.S.)

V/

V/

V

s/

s/

Y

X/

V

V

s/

s/

IO-CHO de-acylase

1 DHFR

H CHO

methylene H 4 reductase * menadione

enzyme RADIOACTIVE ternary complex with X -

5-CH3-H

5,10-CH 2 -H 4

Separation of X - Pte Glu „

4

10-CH0-H,

H2

|DHFR

H j and PteGlu n

Electrophoresis

DetectionQuantitation

Fluorography - Densitometry

Figure 1 (For explanation see text) reduction of PteGlu n .

With high levels of dihydrofolic reductase

the fully oxidized PteGlu n also undergo reduction to H4~PteGlu n and can be quantitatively recovered (37,38).

10-formyl-tetra-

hydrofolates are determined by converting them first to H4"PteGlu n by the use of 10-formyl-tetrahydrofolate deacylase and then to the desired 5,10-CH2-H 4 -PteGlu n by formaldehyde treatment (39).

At

present, no procedures are available for quantitating separately the 5-formyl, 5-formimino and 5,10-methenyl tetrahydrofolate pools.

Because of their recent introduction, these methods have

not yet been extensively applied to the study of natural folates. There is every reason to expect, however, that they will soon become the methods of choice.

452 In-vivo changes in the chain length of the pteroylpoly-Y-glutamates in response to alterations in the steady-state of one-carbon metabolism. There can be little doubt that the requirements for one-carbon transfer reactions must differ, within the same species, from organ to organ. If changes in polyglutamyl chain length are involved in the regulation of these reactions, different patterns of chain length distribution ought to be found in different organs. Using our differential cleavage procedure, we have studied the chain length patterns of total folates in the major organs of the rat and in the liver, kidney, testicle and brain of the Japanese quail. The results are summarized in Table 1. It is clear that pronounced differences do exist confirming and expanding earlier reports (20). The ratio of percent of folates with six and seven glutamyl residues to folates with five residues has been arbitrarily chosen to highlight the differences observed. The spleen and lungs have a predominance of hexa and hepta glutamates with significant amounts of octa detected in both organs. The small intestine presented a unique pattern with a very high proportion of monoglutamates not found in any other organ. These did not come from degraded dietary folates since fasting did not modify the pattern. They may well represent biliary monoglutamates undergoing reabsorption in the entero-hepatic cycle (40,41). It is also very unlikely that this high proportion of monoglutamates is an artifact of degradation by intestinal conjugases. The first step of our procedure inactivates the conjugases instantly by homogenization in 0.1N HC1. Significant organ to organ differences were also seen in the quail. Interspecies differences do exist together with some striking similarities as seen, for example, comparing the testicle folate patterns of the rats and birds. If these differing patterns reflect different one-carbon fluxes through the reactions that compete in the animal organism for the folates of the cell, mainly those involving 5-CH3"H4-PteGlun for the regeneration of S-adenosyl-methionine on the one hand, and 10CH0-H4-PteGlun and 5,10-CH2-H4PteGlun for purine nucleotide and thymidylate synthesis on the other, it should be possible to

453 •o

0) •P

r(0 c td Oi M 0

+ O

o

CO

rH •rH m 3 O'

00

•O c IB 4J (0 u >M 0

co 0) •p (4

e

IB +J 3 rH O" >1 rH 0 0. .H >1

(0

in rH rH

in rn

r~ CN

ro 00 CN CM

cm rCM rH

ai «—t -U-—. 1« IIIS •p f—t 3. 0 o •— MH

-l -U c 0 u

ra

-o ai MH

•


5) ± S.D.

m a i n t a i n p r o t e c t i v e l e v e l s of drug.

E v e n at these h i g h d o s e s , t h e e f f e c t s of

c a r b a m a z e p i n e a n d v a l p r o a t e a r e fairly small. a r e d i s t r i b u t i o n of folate a p p e a r s to o c c u r . i n c r e a s e a s liver levels d e c r e a s e m e n t the p a t t e r n r e v e r s e s .

(Fig. 4).

With chronic valproate

treatment

Initially brain and plasma But a f t e r s e v e r a l w e e k s of

levels treat-

498

11.010.0-

7 =-0.617 p a.

Liver

ï ' T - H

0.6-

0.4. 0.302-

c

o o

7 =-0.454 (NS)

Brain

0.1-

—I

1—

0 06 0.06-

t"T

0.040.030.02-

7=+0.077 (NS)

Plasma

0.01-

-I

1

1

1

1

1

0

1

2

3

4

6

1 6

Weeks of Chronic Phénobarbital Treatment

Figure 2. The effect of chronic phénobarbital treatment on folate concentrations. Dose of phénobarbital was 50 mg/kg. For details see Fig. 1. Folate concentration in liver declined significantly with time of chronic treatment. No significant effects were observed with respect to folate concentrations in brain or plasma. Each point represents the mean (n 5) ± S.D.

Carbamazepine does not seem to cause a folate depletion in any tissue of the rat.

Indeed, the only significant effects of chronic carbamazepine treatment

are the late increases in folate concentration in liver and plasma (Fig. 5).

Discussion There is no consistent pattern of changes in folate concentrations with chronic treatment using different anticonvulsants.

This suggests that the effect of

499

Uvar Folata • -0.682 p«0.06

Brain Folata > = 0.275 INSI

oja»»-

Plasma Folata TV, = 0.666 p< 0.06

Weeks of Treatment with Primidone

Figure 3. The effect of chronic primidone treatment on folate concentrations. Primidone was maintained in homogeneous suspension in water with constant stirring and administered at a dose of 100 mg/kg every 12 hours. Control animals were given water. Rats were sacrificed and folates determined as described in Methods. Means from all treatment groups are significantly different (p 6) ± S.D.

lowering the folate concentration is not a component of the anticonvulsant activity of antiepileptic drugs as some have suggested (15).

Indeed the time

course for the effects on folate concentrations indicates that, where significant effects are found, the effects are slow and gradual when compared to the anticonvulsant effect of the drugs.

In brain, where one would expect to first

observe effects on folate concentrations, if such effects were involved in the

500

W e e k s of Treatment with Valproate F i g u r e 4. T h e e f f e c t of c h r o n i c v a l p r o a t e t r e a t m e n t o n folate c o n c e n t r a t i o n s . V a l p r o i c a c i d w a s d i s s o l v e d in w a t e r at 250 m g / m l b y a d j u s t i n g t h e p H to 7.2 w i t h NaOH. T h i s s o l u t i o n w a s a d m i n i s t e r e d to r a t s e v e r y 8 h o u r s at a dose of 300 m g / k g as d e s c r i b e d in M e t h o d s . T h e c o n t r o l s o l u t i o n w a s 0.96 M N a C l . Rats w e r e s a c r i f i c e d a n d f o l a t e s w e r e d e t e r m i n e d as d e s c r i b e d i n M e t h o d s . Each point r e p r e s e n t s t h e m e a n ± S.D. of 6 - 1 0 a n i m a l s . * Indicates significant difference f r o m c o n t r o l (p

C C

.

D

7

k3

>

D

Equation (1) Equation (2)

where the letters represent: "A"

- non hydrolyzed folate polyglutamate in the lumen

"B"

- hydrolyzed folate polyglutamate (1), or PteGlu (2) in the lumen

"C"

- folate in the intestinal tissue

"D"

- folate transported into the blood

k1, k2, and k3 are the rate constants for these reactions. The rate constants estimated by fitting the experimental data to the theoretical equations are reported in table 2.

Table 2:

Rate Constants Estimated for Equations (1) and (2) equation (2)

equation (1) mean ± SD

1

k1

0.108 ± 0.008 min."

k2

0.046 ± 0.006 min." 1

0.048 ± 0.004 min." 1

k3

0.421 ± 0.044 min." 1

0.189 ± 0.040 min." 1

- -

The fit of the experimental data to the theoretical equations was good in both cases, as shown by the residual sum of square and by the s.d. found for the parameters.

In the case of folate polyglutamates (equation 1),

the rate of hydrolysis (k1) was twice as high as the rate of luminal disappearance of hydrolysis products (k2).

The values for k2, corresponding

to intestinal transport of monoglutamyl folate, were similar in equations (1) and (2).

The higher value for k3 of equation (1), compared to equation

(2), corresponding to blood transport, may reflect delay in the release of absorbed folic acid from the enterocyte.

582 Conclusions We report a simple and convenient method to obtain biosynthetic, purified, radiolabeled folylpolyglutamates.

These polyglutamates can be used as a

tool to study digestion, absorption, and metabolism of natural folate. Data on intestinal absorption are consistent with a model which assumes that hydrolysis to monoglutamyl derivatives precedes transport into the intestine.

Further work is needed to establish the exact location of this

intestinal hydrolysis and the origin of the operating pteroylpolyglutamate hydrolase.

References 1.

Butterworth, C.E., R. Santini and W.B. Frommeyer. 1963. The pteroyl polyglutamate components of American diets as determined by chromatographic fractionation. J.Clin.Invest. 42, 1929-1939

2.

Rose, R.C., A.M. Hoyumpa, Jr., R.H. Allen, H.M. Middleton, L.M. Henderson and I.H. Rosenberg. 1984. Transport and metabolism of water-soluble vitamins in intestine and kidney. Fed Proc, 43 (9), 2423-2429

3.

Selhub, J., 0. Ahmad and I.H. Rosenberg. 1980. Preparation and use of affinity columns with bovine milk folate-binding protein (FBP) covalently linked to sepharose 4B. Methods Enzymol., 66, 686-690

4.

Elsenhans, B., J. Selhub and I.H. Rosenberg, 1980. Assay of folylpolyglutamate hydrolase using pteroyl-labeled substrates and selective short term bacterial uptake for product determination. Methods Enzymol., 66, 663-666

5.

Selhub, J., G.M. Powell, and I.H. Rosenberg, 1984. Intestinal transport of 5-methyltetrahydrofolate. Am J. Physiol., G515-G520

SELECTIVE INHIBITION OF BACTERIAL CARBOXYPEPTIDASE G AND PANCREATIC CONJUGASE BY 2-MERCAPTOMETHYLGLUTARIC ACID

T.I. Kaiman, V.K. Nayak and A.R.V. Reddy Departments of Medicinal Chemistry and Biochemical Pharmacology State University of New York, Buffalo, New York 14260

Introduction The activity of H^folate cofactors and the therapeutic utility of a variety of cytotoxic antifolates may depend on the extent of their polyglutamylation

(1,2).

The biosynthesis and degradation

of the poly-y-glutamate chain is catalyzed by folylpolyglutamate synthetase

(FPGS) and conjugases

22.12), respectively

(2).

(y-glutamyl hydrolases, EC 3.4.

Intracellular regulation of the poly-

glutamate chain length may involve both elongation and cleavage (2,3) by FPGS and conjugases, respectively

(see Fig. 1), but the

role of cellular conjugases in this process has remained elusive. For an effective regulatory function, a likely enzyme candidate with the required hydrolytic activity is a non-lysosomal cytopla smic carboxypeptidase catalyzing the stepwise degradation of a folate polyglutamate chain.

It was of interest, therefore, to

develop selective inhibitors of carboxypeptidases specific for the cleavage of terminal glutamic acid residues.

Such inhibitors may

be useful in the identification and isolation of cytoplasmic conjugases as well as in the study of the metabolism and function of folate and antifolate polyglutamates.

ATP

GLU

+

FPGS

X-H^PTEGLU,

ADP

+

P

X-H^PTEGLU,

N

+

1

(¡¿I CONJUGASE

Figure 1.

Folate Polyglutamate Metabolism.

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

584

Results Our approach to the design of inhibitors of C-terminal glutamate specific carboxypeptidases was analogous to that of Ondetti et al. (4) in obtaining selective inhibitors of carboxypeptidase A and B, which, in turn, was based on the work of Byers and Wolfenden (5). The rationale is outlined in Fig. 2, using carboxypeptidase G (6)

FN7YMFS

/CARBOXYPEPTIDASE A /CARBOXYPEPTIDASE B \ /CARBOXYPEPTIDASE G \

S - C H 2 - C H - coo Figure 2.

Rationale for the Design of Carboxypeptidase Inhibitors.

as the model target enzyme.

Specificity of binding of the inhi-

bitor molecule is provided by the "C-terminal" carboxy group and the carboxyethyl side chain attached to the a-carbon, whereas the presence of the sulfur in the proximity of the active site Znatom provides for the necessary strength of binding for potent inhibitory activity.

The simplest molecule incorporating these

desired structural features is 2-mercaptomethylglutaric acid (MMGA) with the configuration corresponding to L-glutamate (see Fig. 2). The synthesis of D,L-MMGA was accomplished in 3 steps as outlined in Fig. 3.

Michael addition of thiobenzoic acid to 2-methylene-

glutaric acid obtained by acid hydrolysis of 2,4-dicyano-l-butene resulted in a thiolester intermediate, which was aminolyzed to get

585 the desired product in 52% overall yield.

No attempt was made to

resolve the racemic mixture. CN (CH2)2 C=CH 2 CN

H+

COOH

COOH

(CH2)2 OCH2 COOH

PHCOSH ( C H 2 ) 2 ^ HC-CH2-S-CO-Ph COOH

COOH RNH 2

(CH2)2 CH-CH2-SH COOH DA-MHGA

Fig. 3. Scheme for the Synthesis of 2-Mercaptomethylglutaric Acid (MMGA). MMGA was found to be a potent inhibitor of carboxypeptidase G^ (6).

Using folate as substrate and following its conversion to -7 (6), a K^-value of 2.3 x 10 M

pteroate spectrophotometrically was obtained.

In contrast, MMGA was a relatively weak inhibitor -4 of carboxypeptidase A (K. = 1.0 x 10 M) and carboxypeptidase B 1 -4 M) , using hippuryl-L-phenylalanine and hippurvl(K^ = 1.9 x 10 L-arginine as substrates, respectively.

The results are summar-

ized in Table 1. Table 1. Comparison of the Inhibition of Carboxypeptidases by MMGA. Enzyme

(MMGA), pM

Carboxypeptidase A

100

Carboxypeptidase B

190

Carboxypeptidase G^

0. 23

The predicted importance of the SH-group of MMGA for its inhibitory activity against carboxypeptidase G-^ was confirmed by replacement of the -CH 2 SH with a -CH^ group: 2-methylglutaric acid showed no detectable inhibitory activity at a 10,000-fold higher concentration

(3 mM) than that required for 50% inhibition (IC^Q)

by MMGA. A partially purified conjugase preparation from chicken pancreas, assayed at pH 7.2, was found to be strongly inhibited by MMGA. The hexaglutamate derivative of methotrexate

H-NP^-lO-CH^-PteGlUg)

was used as substrate and the formation of the diglutamate endproduct was quantitated by reversed phase HPLC. 6

Using 4.3 x 10

M substrate, an IC50-value of 9.3 x 10~ M was obtained for MMGA.

586

In contrast, no inhibition by 2 x 10 ^ M MMGA of partially purified lysosomal hog kidney conjugase

(7) was observed at pH 4.7.

Conclusions The results demonstrate that selective inhibition of glutamyl carboxypeptidases and yglutamyl hydrolases can be achieved.

A

prototype inhibitor, 2-mercaptomethylglutaric acid, was designed and proved to be effective in selectively interferring with the activities of these enzymes.

This type of enzyme inhibitors can

serve as useful biochemical tools and may also have potential therapeutic applications..

Acknowledgement This work was supported by grant CA 3 5212 awarded by the National Cancer Institute.

References 1.

Goldman, I.D., ed. 1985. Proceedings of the Second Workshop on Folyl and Antifolyl Polyglutamates. Praeger, New York.

2.

McGuire, J.J. and J.K. Coward. 1984. In: Folates and Pterins, Vol. 1 (R.L. Blakely and S.J. Benkovic, eds.). Wiley, New York. pp. 135-190.

3.

Whitehead, V.M. and D.S. Rosenblatt. 1985. Proc. Amer. Assoc. Cancer Res. 26, 232.

4.

Ondetti, M.A., M.E. Condon, J. Reid, E.F. Sabo, H.S. Cheung and D.W. Cushman. 1979. Biochemistry 1427.

5.

Byers, L.D. and R. Wolfenden.

6.

McCullough, J.L., B.A. Chabner and J.R. Bertino. J. Biol. Chem. 2^6, 7207.

7.

Brody, T., J.E. Watson and E.L.R. Stokstad. Biochemistry 2_1, 276 .

1973. Biochemistry 12, 2070.

1982.

1971.

SUMMARY FOLATE ABSORPTION AND TRANSPORT

I.H.

Rosenberg

Clinical

N u t r i t i o n Research

Chicago,

Illinois

transport

at this meeting

progress

Symposium.

to

review

s o r p t i o n and rent work

They project

tumor c e l l s , conceptual transport

Since

of

study:

out o f ,

approaches

1969

predominant

1)

enzymes

intestinal

we

involved

have

forms

to

known

of

in m e m b r a n e

that

in the d i e t ,

of folate

polyglutamyl

w e r e hydrolyzed

is the form which

polyglutamate

the variability

kidney, and

binding

and

is

in the

Only recently h a s the isolation and

ferent s p e c i e s explained

re-

uptake

folates,

circulation.

pteroyl

these

translocation.

folate

intestinal

cur-

technical

specific

and m o n o g l u t a m y

tion of

ab-

dietary

c e l l s as well as in

isolation

of

on three in

to

will

Many of

involved

2 ) the mechanism

7th

I

c o l l e c t i o n of

and h e p a t o c y t e s and 3 ) the evolving

proteins

the

in folate transport.

in this v o l u m e ) by focusing

folate d i g e s t i o n ; even

reflect

the current s t a t e of our u n d e r s t a n d i n g

elsewhere

areas

and

absorption

approaches

(presentations will be cited by authors.

polyglutamyl into,

events

some promising

t r a n s p o r t , based on this exciting

are published lated

on the intestinal

in the s t a t e of k n o w l e d g e since

the study of the molecular attempt

Chicago,

of f o l a t e a c r o s s m a m m a l i a n cell m e m b r a n e s

the substantial Pteridine

of

60637

The paper presented and

C e n t e r , University

intestine

released

into

from

in earlier

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

the

characteriza-

hydrolases seen

the

dif-

studies.

588 Following long

their

report

predicted

in human two

brush border

intestine,

distinct

border,

situated

from

ideally

into

human for

studies

in pig

two

important

as a model

for

from

intestinal

and chick.

PPH's have been purified

to

dietary

folate hydrolysis using

Olson,

Selhub,

lack

an

intrinsic

and Rosenberg

PPH from rat

intestine,

showed

to be the single

this

no evidence of

previously

releasing

in contrast

to

are exopeptidases. neutral

Selhub

by

gestion and

reduced

purified

identifiable

the brush

border

enzyme

enzymes

were

purified

system employing

by

use

These Selhub

immobilized

and Malec's milk

perform

for

group,

and

in the rat

with

as

enzyme

is

a

an

first

and

man

exhibited in-

Darcy-Vri11 on, casei

to

obtain

study of folate po1yg1utamates

affinity

folate binding

by

cytosolic

significantly

L.

"natural"

thus

in pig

zinc.

of

which

monkey)

This enzyme

the rat

pH optima which

dif-

and

the

this

of

absorption,

The study

monog1utamate

Interestingly,

the

by

enzyme.

fo1y1po1yg1utamates

absorption.

promise

PPH and

by

intes-

species from

characterized

pteroyl

reported

The

enzymes.

interaction of the enzyme with

and Rosenberg

biosynthetic

readily

and acidic

PPH from pig

the human and pig

brush border

further

which

this meeting

insights:

secreted

brush

intracellular

(guinea pig, dog,

a second brush border

endopeptidase,

fluenced

the other

in which

isolate

hydrolysis

The only other

appear

both

and

to

the (PPH)

in the

human studies of folate

fer

which

po1yg1utamate

recent

the true species differences the rat,

one

The d e s c r i p t i o n at

and

product,

intestine,

the enterocyte,

hydrolase

proceeded

Wang, and Halsted of a brush border

tine emphasized

identified

pteroylpo1yg1utamate

less c e r t a i n function.

Chandler,

which

C h a r l e s H a l s t e d ' s group

enzymes

precedes uptake with

in Science years ago

diwere

chromatography

proteins.

These

589 should and

help

to establish whether

biochemical

polyglutamates folates.

are

facilitated

fully

and their

greatly

and

similar

to

probes for

as

for

transport

so

and

the

m a m m a l i a n cells. testine

is

acid

the

membrane

controlled

transport

membrane

with similar

affinities for

methotrexate

(in contrast

subsequent

lumenal

reduced

isolation,

intestinal

to transport

folate

important

and

function

is now

uptake

studies

across

that

and

in 1970,

other

this

the folate uptake

into

structure-specific folates

tumor

been

cell

first

represents at

and

lines)

reported

least

three

elucidated

low-outside vs higher and

per-

single

vesicles when pH g r a d i e n t s

ionization

in-

Using

unreduced

surface which have

and

in

this

ionic gradients.

This pH dependence,

A pH gradient, in

compounds

m e m b r a n e s which

to b a c t e r i a and some

studies with membrane

in 1) changes

Some

may become

vesicles was saturable,

laboratory

rigidly controlled.

of

pH and

pH dependent.

p h e n o m e n a at the

carboxypeptidase

with and d i f f e r e n c e s from

demonstrated

brush border

results

of

The newest approach

John B l a i r ' s

Kalman,

in hydrolase enzymes

process

similarities

mit a n a l y s i s of

and strikingly

by

be

PPH's.

the

technique, we previously

hydrolysis

reported

u t i l i z e s v e s i c l e s from brush border

under

biosynthetic of

PPH.

location,

folyl-

Such s t u d i e s will

inhibitors of

pancreatic

study of

and other

clear,

reduced

that

location and species v a r i a t i o n

becoming

from

such

with selective

the further

to

physiological

unreduced

to compare rates

S-mercaptomethylglutaric

intestinal

If the

synthetic

applicable

by work

Reddy

insights from

interrelationship.

which also h a v e activity

of

of

We should now be able

and transport

Nayakj

studies

the

diffusibi1ity

by were

inside of

the

590 transported

species;

(substrate/carrier transport. of folate

2)

affinity);

vesicles

3) c h a n g e s

from

A n n Reisenauer

pig

to

and

transport

unidirectional

transport. other

vesicles

cesium and other

sistent with acids.

to

monovalent affect

the model

Studies

influx

examine

across

Na"

requirement

cations

i.e.

could

the K™ of

of folate uptake.

d a t a from

brush

vesicles from rat

new data sheds

light on

process

enterocytes

from

a

structure which

for

in

and Strum

for

the

intestine. specific,

appears

inconamino

to

the

non-

time,

exciting

saturable be

are

presented

first This

in

Na*.

a K"1" ionophore,

component

and,

folate

guanidium

of Na"* and glucose or

saturable

membranes

jejunal

observed

substitute

Said

dis-

studies

rat

lithium,

a role for monovalent c a t i o n s

border

be

transport, a finding

of cotransport

with

between

the sodium e f f e c t s of -

the

isolated

in v e s i c l e s with v a l i n o m y c i n ,

membrane

pH-dependent

reported

most consistent with

basolateral

border

This relationship

w a s not specific,

Further, Na4" did not

brush

study

transport

Z i m m e r m a n and Selhub

In these studies

preparations

this technique for jejunal

Km

coupled

in m a m m a l i a n cells and tissues will

using

border

folate-H"*

saturable,

to vesicles.

Rosenberg,

brush

transport

c h a r a c t e r i s t i c s of

cussed below. measuring

the of

used

demonstrate

of folate comparing

extent of folate binding binding

in

Three p a p e r s here employed transport.

transport

changes

exit

shared by

all

fo1ates.

The paper Utrecht cells and

by

Schornagel,

shifts our

Rijksen,

attention

emphasizes

methotrexate-resistant

that cells

de

to other, the to

lack

Gier,

and

Jansen

non epithelial of

cross

the antitumor

from

mammalial

resistance

antibiotic

of

CI-920

591 is further contrast

evidence for to

the situation

system e x p l a i n s

Donald H o m e

reported

a

affinity with

system.

Km

Pupons, in

transport

of

the

system

system which

low

affinity

important

reducing

agents.

us that folate bioavailability which

polyglutamyl

measures

folate

shown

demonstrated

liver

to

be a weak

that

study.

Rhee,

that

insight

methotrexate

is impaired by

f u n c t i o n of binding

transport.

most

Selhub, that

and proposed

Emmanouel,

in renal

an

By

Using

work

or carrier Hjelle,

intact

rat yeast

to

acid.

folic

by Zimmerman and

transfer

in

et

Burgert by

mam-

this

in folate

Caroni

provided

and

kidney

and c o n v e r s i o n of

session

proteins

p r o t e i n of rat

a monkey

reminded

deficiency.

new

transport

by

this assay

in vitro

e n d o c y t o s i s as the mechanism

f o 1 a t e - p r o t e i n complex.

by

superior

iron

exciting

the folate binding

is involved

and Lampi

system of folate

addressed

the

is

inhibitor

the

tubule

system

O'Connor, Picciano, Sherman,

the complex

into milk

judged

uptake.

In my opinion,

evidence

this

methodological

Hoppner

c a n be

availability

al , is .not inhibitory.

mary gland

to

is impaired by o x i d a t i o n stress which c a n be avoided

use of natural

Aspirin,

In

is the higher

significance of

studies of h e p a t o c y t e m o n o l a y e r s by showing

bioassay

as

range deserves further an

5-methyl

and folic acid appeared

is pH-dependent

The physiological

and Nimaec provided

in

transport

in isolated hepatocytes.

tetrahydrofolate

in the millimolar

transport the

in tumor cells,

best.

studies

transport

this tissue 5-methyl

of reduced

in intestine where a single

the f i n d i n g s

tetrahydrofolate

share

the carrier

of

proximal folates

translocation of

kidney cell

line

the

in which

592 the folate the

transport

cell,

Kamen

"receptor"

-

and

specificities

returned

testine as well. transport,

novel

contrast

on binding

attention

bind

reductase

to

(DHFR)

folate transport and Rosenberg hibitors be able

to

To study approaches

amined

system

the other

this by synthesizing

in which for

growing

and thus

tissues.

Price,

novel

the

initiate research.

an exciting

in-

participate

in

that

Such drugs

dihydrofolate -

specific Selhub,

three DHFR T h u s one

transport Sams,

in-

should system.

Harpring,

Smith and Fresheim

ex-

photoaffinity

labeling

reagents

Price et al reported

exciting

new

the probe

biology of

new phase

in

data

labeled a ¿»6K p r o t e i n which carrier

the tools

the molecular

in

using

system. even

intestine

Zimmerman,

Fresheim,

by Price,

to Ann

protein

the structure

line of L1210 which

and

pig

inhibit

folate transport.

the transport

that

similar

realization

absent from a mutant cell It appears

kidney

intestine. in

of for

isolation are being employed.

inhibitors as probes of

one by Kempton,

contender

Both

were

the

this conclusively

intestinal

and Kohrs and

a strong

to

inhibitors of

demonstrated

L1210 cells

to

in m a m m a l i a n

based upon methotrexate. on

which

these p r o t e i n s which

the

are also

to use DHFR

Smith,

evidence

folates.

transport

the active center

inhibit

Two reports,

and

other

to the function of the binding

approaches are based upon which

of

specificities in

the folate content

provided

endocytosis

transport

Reisenauer's paper

is regulated by

Capdevila

mediated

studies showed binding

system

to probe these this

protein, lacks the

which

lively

events

proteins

area

was

transport

the molecular

transport

is

of

will

folate

STUDIES

ON

GLYCINE

Wagner*+

Conrad

N-METHYLTRANSFERASE

and R o b e r t

Cook+

J.

VA M e d i c a l Center* and D e p a r t m e n t of B i o c h e m i s t r y + , Vanderbilt U n i v e r s i t y S c h o o l of M e d i c i n e , N a s h v i l l e , T e n n e s s e e , 3 7 2 3 2

Introduction Sarcosine in

is p r o d u c e d

liver

demethylation chondria 1)

methyltransferase accounting

for

from rabbit

(Fig.

1).

direct This

latter

(GNMT;

1.5%

of

protein

5-CHg-H^PteGlUg size

We

which

believe

soluble

an

Fig.

glycine GNMT

allosteric

in

the

nine

levels

the in

"methyl

the

diet

r e g a r d i n g the properties

trap" (3).

in We

the GNMT

of

GNMT

adjusting report

as

bound

of

to

here

to

reaction synthe-

b u t to m o d u -

S-adenosyl-homobe

important

decreased

further

of G N M T a n d i n h i b i t i o n

the

is n o t

( A d o M e t ) to

complementing

cytosol

(1).

a role would

Such

abundant

liver

as F B P - C I I

N-

but not c o v a l e n t l y ,

late the r a t i o of S - a d e n o s y l m e t h i o n i n e liver.

Fig.

by g l y c i n e

has no k n o w n m e t a b o l i c f u n c t i o n ,

in t h e

mito-

7,

identified

inhibitor

role

in the

(reaction

cysteine

(AdoHcy)

1)

choline

oxidative

is e x t r e m e l y

protein

designated

of

the

is c a t a l y z e d

tightly,

the m e t a b o l i c

sarcosine, which

4,

by

GNMT was recently

previously is

that

of

EC 2 . 1 . 1 . 2 0 ) . the

formed

(reaction reaction

a n d 0.73» f r o m r a t .

binding

in the t u r n o v e r

is

methylation

isolated from rat liver c o n t a i n s (2).

It

of d i m e t h y l g l y c i n e

or by the

in the c y t o s o l .

folate

as an i n t e r m e d i a t e

mitochondria

in

methio-

information

by v a r i o u s

folates.

Results Kinetic measurements rate cine.

radioactive

carried out using

T h e pH o p t i m u m of G N M T is a b o u t 9.0 a n d w h e n m e a s u r e d at

this

contrast

obtained

to

values

is 1 . 9 7

from

purified

mM a n d f o r A d o M e t

the

data

obtained

of

0.13

mM

and

by

Ogawa

30 uM

for

AdoMet

sepagly-

glycine

produced

an H P L C m e t h o d to and

p H , the K m f o r in

were

sarcosine

is 78

and

Fujioka

glycine

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

uM. and

This (4)

is who

AdoMet

594

MITOCHONDRIA

CYTOSOL

CHOLINE

©I a ©|

BETAINE-ALDEHYDE BETAINE _

U HOMOCYSTEINE

^{•»METHIONINE

DIMETHYLGLYCINE ^UH^PteGius AdoMet

Ado Hey

•SARCOSINE- •-SARCOSINE

GLYCINE

UHiPteGlus r^SJO-CHj-H^PteGluj^

®

GLYCINE

®\

SERINE

Fig.

1.

Choline

respectively for AdoMet pH

9.0

Hill

turnover

at pH 7 . 4 .

They

plot was

no

evidence

of G N M T

tion

25.7

and w a s

positive

also

observed

positive

cooperativity

for

this.

The

n

value

obtained

at

from

a

1.03. a c t i v i t y was

l o w e d by 5 - C H 3 - H 4 P t e G l u 6 and

liver

at b o t h pH 7.4 a n d 8 . 8 w h e r e a s o u r r e s u l t s c a r r i e d o u t

showed

Inhibition 6.8

in t h e

greatest

and 5 - C H 3 - H 4 P t e G l u 3

pM, r e s p e c t i v e l y .

The

complete

at 2 4 ° .

in 15 m i n

cooperativity

with

for AdoMet

S-CHj-H^PteGlug

w i t h Kj v a l u e s

inhibition

when

required

There

was

measured

fol-

of

4.9,

preincuba-

no e v i d e n c e

in t h e

for

presence

of

5-CH3-H4PteGlu5.

Binding

of r a d i o a c t i v e

unlabeled length

folate

and

important

the

ligands 5-methyl

factors

specificity.

3 6 ( S)5-CH3-H^ [ H]PteGlu5

in

(Table group

binding,

1). on

the

indicating

Both

was

competed

for

polyglutamate

reduced

pterin

a high

degree

ring of

by

chain were

binding

595 Table

1.

of 6 ( S ) 5 - C H 3 - H 4 [ 3 H ] P t e G l u g

Inhibition

Binding

to

Glycine

N-Methyltransferase

Inhibitor

Concentration

Y-GIU-Y-GIU-Y-GIu

1

0 1

1

0

10 100

0

5-CH,-H.PteG1u, 4

1

binding

reaction

and 0 . 0 1 6 the

was

obtained

animals

The

binding

of

studied

using

and

their uM,

tetramer. AdoMet

Human

as

of

8 22 50

1 10 100

1 3 25

out

0.01 by

M

using

HPLC

previously aj[.

from

for

This

is

of

at pH 7 . 0

enzyme for

with

(80%

15 m i n

at

6(S)5-CH,-

GNMT

preparation

[ 3 H ] P t e G l u as

fully

(2).

in t h e Only

consistant

compounds

nanomolar and

one

with

polyglutamates

of t h e s e

5-CH3-H4PteGlug

respectively.

ug

a crude

injected

the f l u o r e s c e n c e

concentration

3

2-mercaptoethanol.

the

of

range.

lack

ligand of

to

GNMT

at pH 2 . 5 The

5-CH3-H4PteGlu3

mole

was

to

dissociawere

0.56

bound

per

cooperativity

for

substrate.

liver GNMT has also been p u r i f i e d

appears Mr

4

1 10 100

two 5 - m e t h y l t e t r a h y d r o f o l a t e

constants 1.6

of

purified

by Wagner £ t

was tion

carried

100

uM 6 ( S ) 5 - C H 3 - H 4 [ H ] P t e G l u g

presence

from

was

0

3

H4[JH]PteG1u5

measure

0

10

PteGlu,

described

0 0

1

J

in

0

10 100

PteGlu,

25"

0

0 0

1

PteGlu,

pure)

Inhibition

1

1

The

%

10 100

5-HCO-H.PteGlu-i

J

(yM)

to c o n s i s t 34,000

of f o u r ,

daltons

each

presumably similar

to

approximately identical, rat

liver

100 f o l d .

subunits GNMT

with

(1,4).

It an The

596 partially

purified

about

same extent

the

purified

rat

human

enzyme

enzyme

is

inhibited

as the r a t e n z y m e . crossreacts

with

by 5 - C H 3 - H 4 P t e G l U g

Antiserum

the

prepared

partially

purified

to

to the

human

enzyme.

Acknowledgement This

work

Veterans to

was

supported

Administration

acknowledge

the

B r i g g s and W a r a p o r n Krumdieck, PteGlUg,

by and

the by

excellent

Medical

NIH

Grant

technical

Decha-Umphai.

We

Research

and

of

We

are

assistance

of

William

also w i s h

to t h a n k

U n i v e r s i t y of A l a b a m a f o r the g e n e r o u s

PteGlUg

Service

IAM15289.

Dr.

g i f t of

the

plesed T.

Carlos

synthetic

PteGlug.

References 1.

Cook,

R.J.

2.

Wagner,

and

Wagner,

C.

(1984)

Proc.

Natl.

Acad.

Sci.

8J,,

3631-3634. C.,

Biophys.

Briggs,

Res.

J.M.

Comm.

W.T.

127,

and W e i r ,

and

Cook,

R.J.

(1985)

Biochem.

746-752.

3.

Scott,

D.G.

4.

O g a w a , H. and F u j i o k a , M . ( 1 9 8 2 ) J. B i o l . C h e m . 2 5 7 ,

(1981)

Lancet

2,

337-340. 3447-3452.

FOLATE BINDING PROTEIN FROM PEDIOCOCCUS CEREVISIAE STRAINS POSSESSING ACTIVE TRANSPORT SYSTEMS FOR FOLATES

Frederika

Mandelbaum-Shavit

D e p a r t m e n t of B a c t e r i o l o g y , Jerusalem, I s r a e l

Hebrew U n i v e r s i t y - H a d a s s a h

Medical

School,

Introduction

We h a v e

shown

previously

cerevisiae/PteGlu,

that

a

s h a r e d by t h e r e d u c e d d e r i v a t i v e s , (MTX),

Pediococcus

p o s s e s s e s an a c t i v e

whereas t h e p a r e n t

cerevisae

a s w e l l a s by t h e a n a l o g ,

their

methyltetrahydrofolate independent indicated

process,

0°C and

P.

the reduced

methotrexate (1,2).

at

only

P.

(PteGlu),

folates

substrates

accumulated

mutant,

system f o r f o l a t e

C e l l s of b o t h s t r a i n s a l s o e x h i b i t e d an a b i l i t y t o p a r t i a l l y a c c u m u l a t e respective

strain

transport

cerevisiae/PteGlu

accumulated

5-

( S - C H j - H ^ P t e G l u ) by a s p e c i f i c and e n e r g y ( g l u c o s e ) not i n h i b i t e d

p r e s e n c e of a f o l a t e

by i o d o a c e t a t e ( 3 ) .

The

latter

data

binder.

B i n d i n g of f o l a t e s by s p e c i f i c b i n d i n g p r o t e i n s p r e s e n t i n many b i o l o g i c a l s o u r c e s h a s been s t u d i e d r e f . 4).

extensively

Membrane-associated

underwent,

as

well,

during the

last

f o l a t e binding protein

detailed

investigation

decade ( f o r r e v i e w from L a c t o b a c i l l u s

which

resulted

in

see casei its

c h a r a c t e r i z a t i o n and p u r i f i c a t i o n ( 5 , 6 ) . The p u r p o s e of t h e p r e s e n t work was t o f u r t h e r e l u c i d a t e f o l a t e b i n d e r a s a component of t h e a c t i v e t r a n s p o r t s y s t e m . strains

varying in t h e i r

transport

properties

made i t more f e a s i b l e t o s t u d y b i n d i n g v s

t h e r o l e of Using

for various f o l a t e

the

bacterial

derivatives

transport.

Results

K i n e t i c s of B i n d i n g of F o l a t e s and t h e E f f e c t of pH S t u d i e s of b i n d i n g of P t e G l u and S-CHg-H^PteGlu by P. c e r e v i s i a e / P t e G l u

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

were

598 performed with energy depleted cells incubated at 5°C. Determination of the time course of binding of PteGlu by 10 9 cells at an external concentration of 10 for 35 min.

juM revealed a maximum binding upon incubation

Binding of S-CH^-H^PteGlu by the same number of cells incubated

at a concentration of 5 jjM achieved steady state within 25 min (not shown). The results depicted in Figs. 1 and 2 show that binding of PteGlu and 5-CHgH^PteGlu follow saturation kinetics and double reciprocal plots of the data (insets) provide affinity constants for this process.

The affinity of the

binder for PteGlu and S-CHg-H^PteGlu is optimal at pH 6.0 and decreases at more alkaline or acidic pH values.

The Kj values for binding of PteGlu (inset

Fig. 1) at pH 6.0, 6.5 and 7.5 were 1.6, 1.9 and 2.7 pH 5.5 the K d was 2.3 jiM (not shown). pH 6.0, 6.5 and 7.0 were 0.9, 1.2 and K d was 1.9 juM (not shown). be 0.28 nmol / 10 cells.

9

The

juM respectively and at

K d values for

5-CH3-H4PteGlu

at

2.4 ,uM respectively, and at pH 5.5 the

The maximal

binding of PteGlu was calculated to

cells, whereas that of 5-CH 3 -H 4 PteGlu was 0.71 nmol / 10 9

The affinity constants for binding of S-CHg-H^PteGlu by P. cerevisiae,

parent strain, determined at pH 6.0 and 7.0 were 0.1 and 0.3 (not shown).

jiM respectively

These cells were unable to bind PteGlu even upon exposure to a

concentration of 50 ^M for 60 min.

Fig. 1

Fig. 2

Figure 1. Binding of folate vs concentration. [2- 14 C] Fol ic acid, potassium salt, sp. act. 52.4 mCi/mmol was purified and quantitated as described previously (2). Growth of bacteria and binding assays: the cells were grown in a Folic Acid Casei Medium, product of Difco, supplemented with folate or folinate 0.4 nM, harvested during the late exponential growth phase and washed with saline. Glucose-depleted cells were obtained by incubation in saline supplemented with 20 mM potassium phosphate buffer, pH 6.5, for 2 hr at 30°C. The cells were collected by centrifugation, washed once in the above solution and suspended (10 cells equivalent to 1 mg dry weight) in 0.95 ml of potassium phosphate buffer 50 mM at the indicated pH. The labelled compound was added in 50 >il and the mixtures were incubated for 35 min at 5°C. The binding was terminated by filtration through membrane filters, 2 washes with

599 i c e - cold s a l i n e and further processed a s described previously (2). Symbols: binding a t pH 6.0 ( • ) ; pH 6.5 (0); pH 7.5 (A). F i g u r e 2. B i n d i n g of 5 - C H 3 - H ^ P t e G 1 u v s c o n c e n t r a t i o n . 5-[1^C] methyltetrahydrofolic acid sp. a c t . 58.3 mCi / mmol. The experimental d e t a i l s were a s f o r F i g . 1, e x c e p t f o r i n c u b a t i o n time which was 25 min. Symbols: binding a t pH 6.0 ( • ) ; pH 6.5 ( 0 ) ; pH 7.0 (A). Binding of F o l a t e s by a Mutant S t r a i n Posessing impaired Transport System for Folates. A mutant s t r a i n of P. c e r e v i s i a e , P. cerevisiae/MTXr, i s o l a t e d by s e l e c t i o n of c e l l s growing in increasing concentrations of MTX exhibited e l e v a t e d a c t i v i t y of d i h y d r o f o l a t e r e d u c t a s e and impaired t r a n s p o r t f o r t h i s a n a l o g and f o r f o l a t e (7).

The r e s u l t s summarized in T a b l e 1 show t h a t the Kd

values

o b t a i n e d f o r binding of v a r i o u s f o l a t e s c o r r e l a t e with t h e i r a p p a r e n t Km values. Table

1.

Kinetics

of

Binding and T r a n s p o r t

of

Various

Folates

in P.

cerevisiae/MTXr and P. c e r e v i s i a e / P t e G l u . P. cerevisiae/MTXr

P. cerevisiae/PteGlu

Km (uM)

Kd (uM)

Km (uM)

Kd (uM)

10.0 a

4.9

6.6a

1.6

5-CH3-H4PteGlu

0.9

0.5

1.2

0.9

MTX

5.0

3.9

0.5

0.2

Compound PteGlu

o For d e t e r m i n a t i o n of t h e K d v a l u e s f o r b i n d i n g of [ H] MTX, t h e P. cerevisiae/MTXr c e l l s were incubated at a concentration range of 1 - 10 uM and P. c e r e v i s i a e / P t e G l u c e l l s a t 0.1 - 1.0 uM f o r 30 min. Other e x p e r i m e n t a l d e t a i l s were a s f o r F i g . 1. a Previously published data (3.7). Binding of Methotrexate and Folate by Plasma Membranes from Pediococcus c e r e v i s a e / PteGlu and Pediococcus c e r e v i s a e / MTXr. The Kj v a l u e s f o r binding of MTX by membranes a r e e s s e n t i a l l y the same a s those obtained with i n t a c t c e l l s .

Thus the a f f i n i t y of the binder for MTX i s

about 20-fold lower in P. cerevisiae/MTXr than that of P. ( T a b l e 1, T a b l e 2).

cerevisiae/PteGlu

S i m i l a r l y , the Kd v a l u e s f o r PteGlu binding by i n t a c t

c e l l s or membranes a r e 3 - f o l d higher in P. c e r e v i s i a e / M T X r a s compared to those of P. c e r e v i s i a e / P t e G l u . Table 2.

Binding of MTX and PteGlu by Membrane Preparations. P. cerevisiae/PteGlu

Compound

Kd (uM)

P. cerevisiae/MTXr Kd (uM)

PteGlu

1.5

4.7

MTX

0.2

3.8

600 Plasma membranes were obtained from lysozyme treated cells by a modification of the method described for Streptococcus cremoris (8): increasing the amount of lysozyme to 1 mg / mg cells (dry weight) and duration of treatment at 30°C to 3 hr instead of 30 min. The binding mixture in 1 ml contained 1 mg membrane protein determined by the method of Lowry (9) in 50 uM potassium phosphate buffer, pH 6.0.

Discussion

Data presented in this study demonstrate that the ability of P. cerevisiae strains to accumulate various folate derivatives is reflected by binding properties of the folate binder.

Thus P. cerevisiae, unable to accumulate

folate could not bind this derivative and an MTX -resistant mutant with impaired transport for MTX due to a 10-fold higher Km value than that of the susceptible strain exhibited an almost

20-fold higher K^ value.

Low binding

affinity for folate has also been reported in an MTX resistant subline of L. casei (10). The affinity of the folate binder for various folates changes with increasing or decreasing pH values in a pattern which correlates with the pH profiles obtained in transport studies of these compounds (2,7). Changes in Kj values with changing pH were also reported in studies of folate binding by L. casei cells (11). In conclusion, the membrane associated folate-binding protein appears to be a crucial component of the active transport system.

Acknowledgment

This research was supported in part by a grant from the Schonbrunn Fund.

References 1.

Mandelbaum-Shavit, F. and N. Grossowicz. 1970. Transport of folinate and related compounds in Pediococcus cerevisiae. J. Baceriol. 104 1-7.

601 2.

M a n d e l b a u m - S h a v i t , F. and N. Grossowicz. 1973. Carrier-mediated transport of folate in a mutant of Pediococcus cerevisae. J. Bacteriol. 114, 485 - 490.

3.

Mandel baum-Shavit, F. and N. Grossowicz. mutant with altered transport of folates.

4.

Wagner, C. 1982. C e l l u l a r f o l a t e binding significance. Ann. Rev. Nutr. 2_, 229 - 248.

5.

Henderson, G.B., E.M. Z e v e l y , and F.M.Huennekens. 1976. Folate transport in Lactobacillus cosei: Solubilization and general properties of the binding protein. Biochem. Biophys. Res. Commun. 68, 712 - 717.

6.

Henderson, G.B., E.M. Z e v e l y , and F.M. Huennekens. 1977. and properties of a membrane-associated folate-binding Lactobacillus cosei. J. Biol. Chem. '252, 3760 - 3765.

7.

M a n d e l b a u m - S h a v i t , F. 1976. Resistance of Pediococcus c e r e v i s a e to amethopterin as a consequence of changes in enzymatic activity and cell permeability. II. P e r m e a b i l i t y changes to amethopterin and other folates in the drug-resistant mutant. Biochim. Biophys. Acta 428, 674 -

1975. Pediococcus cerevisiae J. Bacteriol. 123, 400 - 406. proteins;

function

and

Purification protein from

682.

8.

Otto, R., R.L. L a g e v e e n , H. V e l d k a m p , and W.N. Konings. 1982. Lactate efflux-induced electrical potential in membrane vesicles of Streptococcus cremoris. J. Bacteriol. 149, 733 - 738.

9.

Lowry, O.H., N.J. Rosebrough.A.J. Farr, and R.J. R a n d a l l . 1951. Protein measurements with the Folin phenol reagent. J. Biol Chem. 193, 265 275.

10.

Ananthanarayanan, M., J.M. Kojima and G.B. Henderson. 1984. Structural and functional properties of the folate transport protein from a methotrexate-resistant s u b l i n e of L a c t o b a c i l l u s casei. J. B a c t e r i o l . 158, 202 - 207.

11.

Henderson, G.B. and S. Potuznik. 1982. I r r e v e r s i b l e inhibition of folate transport in Lactobacillus casei by covalent modification of the binding protein with carbodiimide-activated folate. Arch. Biochem. Biophys. 216, 27 - 33.

IMMOBILIZED FOLATE BINDING PROTEIN FROM COW'S MILK USED FOR QUANTITATION OF FOLATE

S.I. Hansen, E. Nex0 Department of Clinical Chemistry, Central Hospital Hiller0d DK-3400 Hiller0d, Denmark J. Holm Department of Clinical Chemistry, Central Hospital Nyk0bing F. DK-4800 Nyk0bing Falster, Denmark

Introduction The purpose of this study was to immobilize the pure folate binding protein (FBP) from cow's milk (1), compare the binding properties of the complex with those of soluble FBP, and use the immobilized FBP for routine determination of erythrocyte folate.

Materials and methods Pure FBP from cow's milk (1) was coupled to microcellulose activais ted with benzoquinone (2) and Dynospheres activated with toluenesulfonylchloride essentially as previously described (3). Binding 125 3 studies were performed with H- and I-labeled folate. Immobilized FBP was incubated with folate for 1 h (25°C, Tris 0.17 M, Triton X-100 1g/1, pH 7.4) prior to centrifugation (3000 g, 4°C, 30 min). Studies with soluble FBP was performed by equilibrium dialysis or charcoal precipitation (4).

Results and Discussion Binding characteristics for FBP insolubilized to cellulose and Dynospheres are compared with those of soluble FBP in table 1. The most important change upon immobilization is the decrease in the apparent affinity constant.

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

604 Table 1 F o l a t e B i n d i n g P r o p e r t i e s of I m m o b i l i z e d a n d S o l u b l e F B P

Solid phase

Cellulose

Y i e l d of c o u p l i n g

%

S t e a d y state for folate b i n d i n g 25°C, p H 7.4

min.

Dynospheres

none

10

10

-

45

60

30 +

D i s s o c i a t i o n of ^ H - f o l a t e a f t e r 1 h a t zero c o n c e n t r a t i o n , 25°C. pH 7 r

pH

- 9

Fraction

3.5

Non specific binding

folate

%

0.1

0.1

0.1 +

>0.8

>0.9

>0 .9 +

-

5+

0

Me

R=H

A; I R = H1

R = cyclopropyl

X. 1 R = cyclopropyl )

Fig. 3 : Generation of A* from DAUM derivatives in acidic media

At reflux temperature (CH^CN/HOAc; ca 80°C) A" could be reduced by either Hantzsch ester (5) or 5,6-dihydrophenanthridine 7 (Fig. 4).

(6) to the thymine derivative

689

O^N^NHMe J. ® •

7o R = H 7b R = 0

O^N^NHMe Me RR EtOOC^YxOOEt

5o R =H 5b R = D

Me^JpMe Fig. 4 : Reduction of A" to 6-ajninothymine derivatives Study of DAUM derivative 4b was expected to throw light on the nature of the reduction step. During the reduction of A1 (R= cyclopropyl), opening of the cyclopropyl ring would imply a radical mechanism involving a SET process, as shown in Fig. 5 ( a - > b - » b ' -»9 ). However, reduction of A' (R= cyclopropyl) by Hantzsch ester (5) and 5,6-dihydrophenanthridine (6) in CH^CN/HOAc at roomtemperature resulted, in both cases, in the formation of "thymine" derivative 8 and not the ring-opened product 9. These results indicate a hydride type mechanism, although operation of a radical mechanism in which intermediate b is quenched by a hydrogen radical, in preference to ring-opening, cannot be excluded at this stage. o

Y

Yr™" O^N^NH, I Me b

O^N^NHj Me b'

0

V

O^-N^nh. J- ©

-

0

Y

!

I Me

N' O^N Me 9

Fig. 5 : Reduction of A' via a radical or hydride type mechanism In view of the observed reduction of intermediate A" by 5,6-dihydrophenanthridine, the folate model 11 was synthesized and examined for its ability to transfer both the methylene group and the hydride equivalent to aminouracil 10•

690 Mi

H

•

0 4-N

-C-Glu

J

11

11

Me

-To Fig. 6 : Chemical modelling of the thymidylate synthase reaction Reaction of 10 with 11 resulted in a mixture in which thymine derivative 7a was identified. Finally, it was also shown that 1_0 reacted with S^O-CH^-H^folate (12) to give spectroscopically identifiable amounts of 7a.

Conclusions The results show that the chemical modelling of the thymidylate synthase reaction, involving both the carbon-transfer and the reduction step has been achieved. Biomimetic studies aimed in particular at the mechanistic details of the reduction step are in progress. Acknowledgements This work was carried out in part under the auspices of the Stichting Scheikundig Onderzoek in Nederland (SON) with the financial support of the Netherlands Organization for Fundamental Research (ZWO).

References 1. Santi,D.V. and P.V. Danenberg.1984.In: Folates and Pterins, Vol.1 (R.L. Blakley and S.J. Benkovic, eds),Wiley, New York, pp 345-398 2. Slieker,L.J. and S.J. Benkovic.1984. J.Am.Chem.Soc. 106, 1833 3. Van der Meij.P.F.C, R.D. Lohmann, E.R. de Waard, T.B.R.A. Chen and U.K. Pandit.1985. J.Chem.Soc,Chem.Comm., 1229

SYNERGISTIC INTERACTION BETWEEN POLINIC ACID AND THE FLUOROPYRIMIDINES

KJiandan Keyomarsi and Richard G. Moran Department of Biochemistry, School of Medicine, University of Southern California, Los Angeles, California 90033, and Children's Hospital of Los Angeles, Division of Hematology-Oncology 4650 Sunset Boulevard, Los Angeles, California 90054

INTRODUCTION The fluorinated pyrimidines have been used extensively in the treatment of certain types of cancer since they were first synthesized in 1957 (1). Exposure to 5-FU or FUdR results in inhibition of the growth of cell lines derived from both solid tumors and leukemias (2) . The effects of the fluoropyrimidines against tumor cells in culture and the related chemotherapeutic activity of 5-FU and FUdR in vivo have been ascribed to three major mechanisms: a) metabolism to FdUMP, which is a potent suicide inhibitor of thymidylate synthase (TS) (3) , b) incorporation of the ribonucleotide triphosphate of 5-FU into some species of RNA (4) , and (c) incorporation into DNA (5) . The inhibition of TS by FdUMP involves the formation of a ternary complex in which the enzyme is covalently linked to the nucleotide substrate which is, in turn, covalently bound to the folate cofactor, 5,10-methylene tetrahydrofolate (5,10-CH2H4PteGlu) (3). In the absence of the cofactor, FdUMP forms a weak binary complex with TS (Kd = 10 "*M) , where as the presence of 5,10-CH_H.PteGlu results in a covalent -9 ternary complex in which FdUMP is bound much more tightly (Kd = 1 0 -12

10 M). Fran this it follows that intracellular levels of reduced folate cofactors could be a critical determinant of the cytotoxic effects of 5-FU and FUdR. In this study, we show a synergism between folinic acid and fluoropyrimidines in leukemic cells.

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

692 RESULTS The growth inhibitory and cytotoxic effects of fluoropyrimidines on leukemic cells of mouse and human origin in the presence of folinic acid were studied (6). Exposure of exponentially growing mouse L1210 cells to low concentrations of FUdR resulted in an initial inhibition of the growth of these cells (Figure 1) . Subsequent recovery of the growh rate of growth of these cells after 12 hours approached that of control cultures, in spite of the continued presence of drug. On the other hand when folinic acid was added to the cultures at the sams time as FUdR, the initial growth inhibition was maintained.

The growth inhibitory effects of 5-FU were 1.5- to 2.1- fold

more potent in the presence of folinic acid while the potency of FUdR was increased by 2.4- to 4.5- fold in the cell lines studied. Furthermore, L1210 cells that were exposed to folinic acid and concentrations of 5-FU or FUdR that were individually noncytotoxic resulted in a 98% to 99.9% cell kill.

Time

hours

Figure 1 Inhibition of growth of L1210 cells by FUdR and folinic acid. Triplicate suspension cultures (20 ml were exposed to PBS (0) , 10

M

folinic acid (•) , 3.2 x 10~10M FUdR (D), or 10-5M folinic acid and 3.2x10 ^ M FUdR ( • ) . Aliquots of each culture were removed at the indicated times and the culture density of each was determined electronically.

Variation among replicates was less than the size of the

symbols. The ordinate expresses the culture density relative to the culture 4 density at time zero (2.5x10 cells/ml).

693

DISCUSSION The most likely mechanism for the synergism between high concentrations of folinate and the fluoropyrimidines is the stabilization of catalytically inactive complexes between TS, FdUMP, and 5,1O-CH^H^PteGlu. Daneriberg and his colleagues (7) have reported that mainrialian TS follows an ordered sequential reaction mechanism with dUMP binding first (Fig. 2) and that formation of a ternary complex with FdUMP and cofactor follows the same sequence of addition of reactants to the enzyme. This order of addition of reactants furnishes an explanation of the folinate induced synergism. S-CHO-H4PtaQlu • FdUMP

I

dCMP^^^dUMPTS I UOP'

5.10-CH2-H,Pt»Glu

' TS.FdUMPMM^» T8.FdUMP.5.10-CHi-HJ 4 DTT 4.30 100 + Met synthase 10.7-7.6 , +catalose 3.55 0

0

2

4

6

8 10 12 TIME (min)

14

16

18

20

22

Figure 2. Characterization of the thiol oxidase activity associated with partially purified methionine synthase from pig liver. The enzyme used for these studies had a specific activity of 0.05 pmoles min~l mg~l, and had been purified by ammonium sulfate fractionation and chromatography on DEAE cellulose and hydroxylapatite (BioRad HTP). Thiol oxidase activity was measured in an oxygen electrode at 37°C. Reactions were measured in solutions containing 25 mM dithiothreitol in 50 mM potassium phosphate buffer, pH 7.2. confirming the oxidation state of the inactive enzyme.

Activation

of the enzyme using the R/F/NADPH reducing system and AdoMet leads to the absorbance changes expected for conversion of cob(II)alamin to methylcobalamin, as previously reported by Fujii and Huennekens (13) and to the disappearance of the cob(II)alamin EPR spectrum. [The copper associated with the enzyme preparation is EPR silent, and can only be visualized as C u + + after denaturation of the enzyme in the presence of mersalyl, urea and EDTA.] Activation of the enzyme is generally postulated

to proceed by

reduction of the enzyme-bound cobalamin to the cob(I)alamin oxidation state with trapping of this species by methylation with AdoMet.

The unliganded cob(II)alamin/cob(I)alamin couple has a redox

potential at pH 7 of -0.61 V vs the standard hydrogen electrode and the potential is -0.50 V at pH 2, where cob(II)alamin exists

704 in the b a s e - o f f c o n f o r m a t i o n due to p r o t o n a t i o n of the d i m e t h y l benzimidazole substituent

(21).

It is p o s s i b l e that the m e t h i o -

nine s y n t h a s e a p o e n z y m e s i g n i f i c a n t l y a l t e r s the redox

potential

of the enzyme b o u n d c o b ( I I ) a l a m i n , b u t c o b ( I ) a l a m i n is n e v e r observed during reductive activation.

We m u s t t h e n e x p l a i n h o w

s y s t e m s like R / F / N A D P H or d i t h i o t h r e i t o l , w i t h redox p o t e n t i a l s the range from - 0 . 3 8 to - 0 . 2 0 V , are able to a c c o m p l i s h of c o b a l a m i n .

in

reduction

It is not c l e a r that m e t h y l a t i o n by A d o M e t can p r o -

v i d e m u c h d r i v i n g force for the r e a c t i o n , since the weak bond in the s u l f o n i u m will be r e p l a c e d by a weak

S-methyl

carbon-cobalt

bond o n the e n z y m e . I think we need to a d d r e s s s e r i o u s l y the p o s s i b i l i t y

that

c o b ( I I ) a l a m i n reacts w i t h A d o M e t by h o m o l y t i c m e t h y l t r a n s f e r , that the r e d u c i n g s y s t e m is r e q u i r e d for the s u b s e q u e n t of A d o H C y , as s h o w n by e q u a t i o n

[4] and

I

+

S

CH-?-cobalamin +

le~ +

/\ Spectroelectrochemical

reduction

[5]

CH3 cob( I I ) a l a m i n + J5

and

•+ S

[4]

S

[5]

/\

t i t r a t i o n s s h o u l d e s t a b l i s h the n u m b e r of

e l e c t r o n s a c t u a l l y r e q u i r e d for a c t i v a t i o n and the p o t e n t i a l at w h i c h they are

approximate

delivered.

A final m e c h a n i s t i c q u e s t i o n c u r r e n t l y of g r e a t interest to m a n y of the p a r t i c i p a n t s

in this S y m p o s i u m c o n c e r n s the m e c h a n i s m of

i n a c t i v a t i o n of m e t h i o n i n e s y n t h a s e by N 2 O , and its ramifications.

Early i n v e s t i g a t i o n s

physiological

(22-24) n o t e d the

development

of m e g a l o b l a s t i c a n e m i a in p a t i e n t s w h o had b e e n e x p o s e d to N2O for p r o l o n g e d p e r i o d s , and the n e u r o l o g i c a l s y m p t o m s of

pernicious

a n e m i a in d e n t i s t s and o t h e r s s u b j e c t e d to c h r o n i c e x p o s u r e N2O.

to

T h e s e s y m p t o m s were c o r r e l a t e d w i t h i n a c t i v a t i o n of m e t h i o -

nine s y n t h a s e in a n i m a l s e x p o s e d to N2O (25-27).

Model

studies

have s u g g e s t e d that c o b ( I ) a l a m i n , a p r o p o s e d i n t e r m e d i a t e m e t h y l t r a n s f e r by m e t h i o n i n e s y n t h a s e , reacts w i t h N2O to e q u a t i o n

[6]

(28,29).

in

according

705 cob(I)alamin + N2O + cob(III)alamin + N2 + H2O

[6]

We began by establishing that methionine synthase from either _E. col i or pig liver can be inactivated by N2O _i_n vitro [Frasca e_t al., this volume].

Inactivation occurs only during turnover and

requires ~3900 turnovers per inactivation event.

Since the

products of equation [6] are inocuous, it was not clear why the reaction of N2O with methionine synthase should result in the irreversible inactivation of the enzyme.

Our studies suggest that

methionine synthase-mediated cleavage of N2O proceeds according to equation [7]. cob(I)alamin + N2O •* cob(II)alamin + N2 + OH.

[7]

We postulate that the generation of hydroxyl radical at the active site is responsible for the irreversible loss of activity, and for the observed partial loss of the cobalamin prosthetic group. Indeed, Kondo and his colleagues (27) reported the accumulation of unidentified cobalamin analogues following exposure of rats to N 2 0. Since N2O remains the anesthetic of choice for many surgical procedures, we need to consider the implications of these findings for patient management following long term exposure to the gas. O'Sullivan and colleagues have already shown that prior administration of 5-HCO-H4folate prevents the development of bone marrow abnormalities on prolonged exposure to N2O (24).

The possibility

remains however, that the cobalamin pools in the body become deficient in methyl cobalamin, and that significant levels of cobalamin analogues accumulate, as in the studies by Kondo e_t a_l on rats exposed to N2O (27). It is clear that nitrous oxide is proving to be an important research tool for exploring folate metabolism _i_n vitro and _i_n vivo. One may note particularly the elegant demonstration by Scott and his colleagues that chronic administration of N2O to monkeys results in development of the neurological symptoms characteristic of pernicious anemia and that dietary supplementation with methionine prevents the development of these symptoms (30).

Rosenblatt

706 and his c o l l e a g u e s

(20) h a v e used N2O to a s s e s s the a c t i v i t y of

m e t h i o n i n e s y n t h a s e inside f i b r o b l a s t s , since t u r n o v e r is for i n a c t i v a t i o n by N 2 O .

required

We a n t i c i p a t e a c o n t i n u e d i n t e r e s t

the use of N2O as a p r o b e of folate m e t a b o l i s m , and

continued

e l u c i d a t i o n of the c o m p l e x m e t a b o l i c s e q u e l l a e of m e t h i o n i n e thase

in syn-

inactivation.

Acknowledgements R e s e a r c h in the a u t h o r s ' l a b o r a t o r y w a s f u n d e d by the N a t i o n a l I n s t i t u t e s of H e a l t h G r a n t

GM24908.

References 1.

D a u b n e r , S . C . , R.G. M a t t h e w s .

2.

C l a r k , J . E . , L.G. L j u n g d a h l . 10845.

3.

K u t z b a c h , C., E.L.R. S t o k s t a d . 250, 459.

4.

M a t t h e w s , R . G . , M.A. V a n o n i , J . F . H a i n f e l d , J . W a l l . J . B i o l . C h e m . 259, 11647.

5.

S u m n e r , J . , D.A. J e n c k s , S. K h a n i , R.G. M a t t h e w s . J . B i o l . C h e m . 261, 7697.

6.

Mudd, S . H . , B.W. U h l e n d o r f , J . M . F r e e m a n , J . D . F i n k e l s t e i n , V.E. S h i h . 1972. Biochem.Biophys.Res.Comm. 46, 905.

7.

Erbe, R.W.

8.

R o s e n b l a t t , D . S . , B.A. Cooper. 1979. In: Folic A c i d in N e u r o l o g y , P s y c h i a t r y and Internal M e d i c i n e (M.I. B o t e z and E.H. R e y n o l d s , eds.). R a v e n P r e s s , N . Y . , p. 385.

9.

N a r i s a w a , K. 1979. In: F o l i c A c i d in N e u r o l o g y , P s y c h i a t r y and I n t e r n a l M e d i c i n e (M.I. Botez and E.H. R e y n o l d s , e d s . ) . R a v e n P r e s s , N . Y . , p. 391.

1975.

1982.

J.Biol.Chem.

1984.

N.Engl.J.Med.

J.Biol.Chem.

1971.

257,

140.

259,

Biochim.Biophys.Acta 1984.

1986.

293, 753, 807.

10.

L e v i t t , M., P.F. N i x o n , J. P i n c u s , J . R . Bertino. J . C l i n . I n v e s t . 50, 1301.

11.

M a t t h e w s , R . G . , S. Kaufman.

12.

T a y l o r , R.T. 1982. In: B12, V o l u m e 2 (D. D o l p h i n , W i l e y - I n t e r s c i e n c e , N . Y . , p. 307.

ed.)

13.

Fujii, K . , F.M. H u e n n e k e n s .

Aspects

1980.

1979.

J.Biol.Chem.

In:

1970. 255,

Biochemical

6014.

707 of N u t r i t i o n (K. Y a g i , ed. ) J a p a n S c i e n t i f i c S o c i e t i e s T o k y o , p. 173.

Press,

14.

M a t t h e w s , R.G. 1984. In: F o l a t e s and P t e r i n s , Vol. _1, (R.L. B l a k l e y and S.J. B e n k o v i c , eds.) J o h n W i l e y , N.Y. , p. 497.

15.

Z y d o w s k y , T . M . , L.F. C o u r t n e y , V. F r a s c a , K. K o b a y a s h i , H. S h i m u z u , L.-D. Yuen, R.G. M a t t h e w s , S.J. B e n k o v i c , H.G. Floss. 1986. J . A m . C h e m . S o c . 108, in p r e s s .

16.

T a y l o r , R . T . , H. W e i s s b a c h . 123, 109.

1968.

Arch.Biochem.Biophys.

17.

F u j i i , K . , F.M. H u e n n e k e n s .

1974.

J.Biol.Chem.

18.

U t l e y , C . S . , P.D; M a r c e l l , R.H. A l l e n , A . C . A n t o n y , J.F. Kolhouse. 1985. J . B i o l . C h e m . 260, 13656.

19.

S c h u h , S . , D.S. R o s e n b l a t t , B.A. C o o p e r , M . - L . S c h r o e d e r , A.J. B i s h o p , L.E. S e a r g e a n t , J.C. H a w o r t h . 1984. N e w . E n g l . J . M e d . 310, 686.

20.

R o s e n b l a t t , D . S . , B.A. C o o p e r , A . P o t t i e r , H. L u e - S h i n g , Mat iaszuk, K. G r a u e r . 1984. J. CI in. Invest. 2149.

21.

L e x a , D. and S a v e a n t , J.M. 1976.

22.

L a s s e n , H . C . A . , E. H e n r i c k s o n , F. N e u k i r c h , H.S. 1956. L a n c e t j., 527.

23.

A m e s s , J . A . L . , G . M . R e e s , J.F. B u r m a n , D.G. D.L. M o l l i n . 1978. L a n c e t i_i, 339.

24.

O ' S u l l i v a n , H., F. J e n n i n g s , K. -Ward, S. M c C a n n , J . M . D.G. W e i r . 1981. A n e s t h e s i o l o g y ^ 5 , 645.

25.

D e a c o n , R . , J. P e r r y , M. Lumb, I. C h a n a r i n , B. M i n t y , M.J. H a l s e y , J.F. Nun. 1978. Lancet 1023.

26.

K o b l i n , D . D . , J . E . W a t s o n , J . E . D e a d y , E.L.R. S t o k s t a d , Eger. 1981. A n e s t h e s i o l o g y 54, 318.

27.

K o n d o , H . , M . L . O s b o r n e , J . F . K o l h o u s e , M.J. B i n d e r , P o d e l l , C.S. U t l e y , R.S. A b r a m s , R.H. A l l e n . 1981. J. Clin. Invest. 6J_, 1270.

28.

B a n k s , R . G . S . , H e n d e r s o n , R . J . , P r a t t , J.M. J . C h e m . S o c . (A), 2886.

29.

B l a c k b u r n , R., M. K y a w , A.J. S w a l l o w . F a r a d a y T r a n s . 7_3, 250.

30.

S c o t t , J . M . , J.J. Dinn, P. W i l s o n , D.G. W e i r , 1981. i, 334.

J.Am.Chem.Soc.

1977.

249,

6745.

N.

9j3, 2652. Kristensen.

Nancekievi11, Scott,

E.I.

E.R.

1968. J.Chem.Soc. Lancet

IMPAIRED FORMYLATION AND UPTAKE OF TETRAHYDROFOLATE BY RAT SMALL GUT FOLLOWING COBALAMIN INACTIVATION

Janet Perry, Rosemary Deacon, M. Lumb and I. Chanarin Medical Research Council, Clinical Research Centre, Northwick Park Hospital, Harrow, Middlesex, U.K.

Introduction

During absorption reduced folate analogues are converted into methyltetrahydrofolate in the enterocyte.

In the case of tetrahydrofolate(H4PteGlu) there is

addition of a formate(CHO-) group which is further reduced to methyl (1). It has been suggested that the role of cobalamin is to make formate units available and that methionine is an important source of such formate units (2). Nitrous oxide (N2O) oxidizes and inactivates cob[I]alamin, the cofactor in the methionine synthetase reaction.

Many of the effects are reversed when formyltetrahydro-

folate is supplied (3). The defect is also reversed when methionine and some of its metabolic products are given with H4PteGlu (4).

Small gut segments in

vitro are able to formylate tetrahydrofolate and hence provide an opportunity for measuring the formylation step directly in control and cobalamin-inactivated (N20-exposed) animals. In view of uncertainty about the presence of methionine synthetase in small gut (5,6), this enzyme was assayed in small gut segments.

Methods

Animals:

Male Sprague-Dawley rats, 80-100g, were used.

Test animals breathed

N20:02 (1:1) in a chamber in which CO2 and humidity were controlled, with free access to commercial rat diet and water.

Preparation of everted gut sacs:

Control animals breathed air.

These were prepared according to Wilson &

Wiseman (7). The test compounds (16umols), together with 2pCi

[2- 1 4 C]H4PteGlu

and 1% ascorbate, pH 6.1 were added to the mucosal fluid, the flasks flushed with 02:C02 (95:5) for 3 minutes and incubated for one hour at 37°.

The

serosal fluid was then drained from the gut sac and retained, and the gut sac,

Chemistry and Biology of Pteridines 1986 © 1986 Walter d e Gruyter & Co., Berlin • New York - Printed in Germany

710

washed with saline, was homogenized in 0.1M potassium phosphate buffer pH 5.7 containing 0.2% ascorbate.

Portions of both samples were counted for

radioactivity to determine total uptake of [ 2 - 1 4 C ] H 4 P t e G l u .

Separation and identification of folates: This has been described elsewhere (4).

Methionine synthetase was assayed by the method of Kamely et al., (8) and protein content measured as described by Lowry et al., (9).

Expression of results: The proportion of mucosal fluid [ 2 - u C ] H 4 P t e G l u metabolized to 1-carbon-substituted folate compounds during absorption was derived from the chromatograms of gut and serosal fluid, and the radioactivity of each folate expressed as a percentage of the total mucosal [2-^CJltyPteGlu.

Statis-

tical comparison between groups were performed according to Hill and Peto (10).

Results

Methionine synthetase activity:

Considerable methionine synthetase activity was

present in whole gut which had been washed through with saline, everted and washed again. Activity was present in all but 2 of 14 specimens from air-breathing rats with assay values from 0.95 to 2.31 nmol methionine protein/hour.

formed/mg

There was no detectable methionine synthetase activity in gut

segments from N20-breathing rats regardless of the method of preparation.

Folate uptake by gut segments.

The mean folate uptake by gut sacs from control

animals was 4.39% of the amount added to the incubation fluid.

There was no

change after 24 hour N2O exposure but a significant fall to 2.51% after 7 days N2O.

The proportion of radiolabelled formylfolate formed from mucosal

[2-14C] PteGlu by control tissue was 1.06%.

This was halved to 0.56% after

24 hour N2O and fell further to 0.26% after 7 days N2O (Figure). differences were significant (p = 25

enzyme from inactivation by proteases (11), suggesting that it induces a conformational change in the protein or holds the protein in a specific conformation.

The d i v a l e n t cation require-

ment is to generate the substrate MgATP, and free ATP is a potent inhibitor of the reaction.

The proteins are monomeric and have

similar turnover numbers with their preferred folate substrates (3,4,12).

The CQtyQeijasteiiuiD and EA c q I a proteins also possess

dihydrofolate synthetase activity, while enzyme from

sa££i
>

methylene-THF (glu-2)

dihydrofolate

tetrahydrofolate

(glu-2)

»

(glu-3,4)

methylene-THP

The physiological relevance, if any, of the change in oxidation state and one carbon substitution of the pteroyl substrate as the glutamate chain is extended is not known.

Each of the indicated

steps requires the involvement of an additional enzyme viz. dihydrofolate reductase and serine hydroxymethyltransferase an additional glutamate moiety can be added.

before

The purified

£o£yn£bac££riuQi enzyme metabolizes folates to the tetraglutamate derivative, which are the types of folates that predominate io 2ÏXQ.

The LâStQfcàÇÀlllJS and IL e e l i enzymes w i l l metabolize

folates ¿q yitr.e to tetra- and triglutamate derivatives, respectively, although longer polyglutamate derivatives are observed ¿q YÀÏQ.

This apparent lack of fidelity, with the Ej.

ç o l i e n z y m e at l e a s t , is due to the p r e s e n c e of an a d d i t i o n a l enzyme in SL s s l i that converts pteroyltri-y-glutamates to long chain polyglutamate derivatives, with the additional glutamate residues added in a-linkage (14). The properties of several mammalian folylpolyglutamate synthetases have been examined (7-10).

The pig liver enzyme is the only

mammalian enzyme to have been purified to homogeneity (5) and its

722 Table 2

General Properties of Pig Liver Folylpolyglutamate Synthetase

Purification (fold) pH optimum monovalent cation divalent cation reducing agent M r (SDS gel elec) M r (Sephadex) DHF synthetase act. -1 cat (min )

40-200,000 9.4 2 0mM K + M g 2 \ Mn22 ++ DTT, ßME 62,000 66,000

NO 127

general properties, which in most regards are qualitatively identical to the properties reported for other mammalian enzymes, are listed in Table 2. The pig liver enzyme resembles the bacterial synthetases in that it is a monomeric protein present at low cellular concentrations (10-50 nM), it has a high pH optimum, and requires mono- and divalent cations for activity, although the monovalent cation requirement is met by lower concentrations. Mammalian synthetases differ from the bacterial enzymes in their absolute requirement for a reducing agent, their lack of dihydrofolate synthetase activity, and by a marked difference in folate substrate specificity (described below). The high pH optimum of folylpolyglutamate synthetases is due a high K m value for glutamate at physiological pH rather than an effect of pH on V m a x . Under physiological conditions, the glutamate concentration in mammalian tissues would not be saturating, suggesting that any modulation of cellular glutamate concentrations would be expected to modulate the rate of folylpolyglutamate synthesis.

723

Table 3

Kinetic Constants of Hog Liver Folylpolyglutamate Synthetase

Substrate H4PteGlu H 4 PteGlu 2 H 4 PteGlu 3 H 4 PteGlu 4 H 4 PteGlu 5 H 4 PteGlu 6 H 4 PteGlu ? HjPteGlu H 2 PteGlU 2

V "

M

7.7 3.4 1.1 2.0 2.7

5.0 2.6

>

K

i("M>

v

max(rel)

100

45

102

8.8

62

11

4.5

14 34 47

1.6

17 5 2000

1

2,5-di ami nopentanoate

2.5

3.2

2,4-diami nobutanoate

5.7

330

2,3-diami nopropanoate

17

glutamate (MTX)

Rat Liver

K

50

nM

R= K562

FPGS Inhibition

1

2,5-di ami nopentanoate 2,4-di ami nobutanoate 2,3-di ami nopropanoate

showed the pentanoate length gives maximum FPGS inhibition.

3.8 200 >1000

The analogs were

relatively weak inhibitors of human leukemia cell growth (Table 2). cytotoxic, the pentanoate MTX itself.

The most

derivative, was 70 to 100-fold less cytotoxic than

However, a CCRF-CEM subline resistant to MTX (250-fold) via defec-

tive MTX transport was only 5-fold cross-resistant to the pentanoate

analog.

This result suggested that this analog might use a pathway of uptake different from that of MTX.

Transport of the pentanoate analog was examined by characterizing its inhibi3 tion of [ H]MTX transport. In these studies, 20 uM pentanoate analog 3 affected neither the initial velocity of transport of 2 nM [ H]MTX by CCRF3 CEM cells nor the plateau level of [ H]MTX at 30 min. Thus, the pentanoate analog either uses a pathway separate from the reduced folate/MTX or uses this transporter with a very low affinity.

transporter

731 Table 2. Cytotoxic e f f e c t s on K562 and CCRF-CEM human leukemia cell

lines

of

2, u-diaminoalkanoic acid analogs of MTX Cell

Inhibitor

line

4-NH 2 -10-CH 3 -Pte-R

EC

R= K562

CCRF-CEM

An analog of f o l i c

50 IIM

2 , 5 - d i ami nopentanoate

1.7

2,4-di ami nobutanoa te

2.3

2,3-diami nopropanoate

3.0

glutamate (MTX)

0.0175

2 , 5 - d i ami nopentanoate

0.74

glutamate (MTX)

0.011

acid,

10-CH 3 -Pte-(2,5-diaminopentanoate)

was also

synthe-

sized.

This compound was a weaker FPGS i n h i b i t o r

than the corresponding MTX

analog

(K^ s >100

state

inhibition

(Kis

pM).

= 7 nM).

reduced d e r i v a t i v e s folates

as

Reduction The

paralleled

substrates.

Thus,

to

the

potency

dihydro

increased

of

inhibition

by

the

the r e l a t i v e

affinities

of

the

the

structural

specificity

of

the

oxidized

FPGS and

corresponding FPGS

for

the

pteridine moiety i s not altered by the s u b s t i t u t i o n of 2,5-diaminopentanoate. These studies indicate that i n h i b i t i o n of FPGS by those compounds i s for the 2,5-diaminopentanoate d e r i v a t i v e . unaltered by t h i s

substitution.

greatest

S p e c i f i c i t y for the pteridine seems

Provided the poor transport

characteristics

of t h i s i n h i b i t o r can be overcome, these studies may allow the design and synt h e s i s of s p e c i f i c FPGS i n h i b i t o r s which may be useful agents.

732 Acknowledgement

This work was supported by ACS Grant CH-288 and CA25236 from the NCI.

JJM i s

a Scholar of the Leukemia Society of America.

References

1.

McBurney, M.W. and Whitmore, G.F. 1974. I s o l a t i o n and Biochemical Charact e r i z a t i o n of Folate D e f i c i e n t Mutants of Chinese Hamster C e l l s . Cell 2: 173-182.

2.

Cichowicz, D., Cook, J . , George, S. and Shane, B. 1985. I n : Proceedings of the Second Workshop on Folyl and A n t i f o l y l Polyglutamates. Hog L i v e r Folylpolyglutamate Synthetase: Substrate S p e c i f i c i t y and Regulation. (Ed. 1.0. Goldman) pp 7-13. Praeger, NY.

3.

Piper, J . R . , McCaleb, G.S., Montgomery, J . A . , Schmid, F.A., and Sirotnak, F.M. 1985. Syntheses and Evaluation as A n t i f o l a t e s of MTX Analogues Derived from 2,0mega-diaminoalkanoic a c i d s . J.Med. Chem. 28: 1016-1025.

AN UPSTREAM GENE REGULATES THE EXPRESSION OF FOLYLPOLYGLUTAMATE SYNTHETASE-DIHYDROFOLATE SYNTHETASE JN ESCHERICHIA COLI

Andrew L. Bognar Department of Microbiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8 Charles Osborne, Barry Shane Department of Nutritional Sciences, University of California, Berkeley, Berkeley, California 94720

Introduction We have recently reported the isolation and cloning of the gene for folylpolyglutamate synthetase-dihydrofolate syntnetase (folC) from Escherichia coli (1).

The folC gene was localized on a 1.5 kb Pvu

I fragment which could complement the methionine auxotrophy of the FPGS-

mutant, SF4 (2).

The smallest DNA fragment whicn also

resulted in amplified expression of the FPGS enzyme when cloned into high copy number plasmids was a 3.5 kb Sst II fragment.

In

this report we describe studies of the expression of FPGS/DHFS activity in plasmids containing the cloned folC gene and snow that an intact upstream gene is required for the high expression of tne enzyme from high copy number plasmids.

Results We have cloned the folC gene by selecting for transformants of plasmids containing E. coli sequences which complement the SF4 mutation.

In the process of subcloning the folC gene, some trans-

formants were found which could complement the SF4 mutant but nad less than wild type levels of FPGS activity in a mutant background. A lOkb Hind III fragment cloned in pKC7 (pH2al, Table I) produces amplified enzyme activity while its suoclone cut with Eco RI (pHE2a4) had only wild type activity.

A 3.5 kb Sst II fragment

was the smallest subclone of the Hind III fragment which gave amp-

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

734 lified enzyme activity when cloned into high copy number plasmids Subclones in which insert DNA was cut at a Kpn I

(pAC3, p2S2-4).

(pAC5R) or PvU I site (p2P2-7) had low activity.

These same sub-

cloned fragments placed downstream from a promoter from the vector, such as the lac promoter (pAC5) of the lambda P T promoter

(p2P2-7)

produced highly amplified enzyme levels in transformants.

These

results initially suggested that the promoter of the folC gene was being deleted by removal of the sequences upstream of these restriction sites. Table 1

FPGS Activities of Transformants of Various Plasmids

Strain

Plasmid

Vector

Site of Deletion

Relative Activity

W1485

-

-

-

1

SF4

-

-

-

0 .09 .

SF4

pH2al

pKC 7

SF4

pHE2a4

pKC7

SF4

pAC3

pUC 8

SF4

pAC5R

pUC8

Kpn I

SF4

a

pUC9

Kpn I

pAC5

-

Eco RI -

SF4

p2S2-4

b

pHE2

SF4

p2P2-7 b

pHE2

Pvu I

SF4

p2P2-7 b

pHE2

Pvu I

a

-

46 1.

.4

71 0 .67 . 15 60 0 .

154

(30°) , 4 (30°) (42°)

The folC gene is downstream from the lac promoter of pUC9 in the proper orientation for transcription.

k These plasmids have the folC gene downstream of the lambda PLi promoter controlled by a temperature sensitive repressor. The DNA sequence of 2.5 kb of E. coli DNA containing the folC gene has been determined.

The Eco RI, Pvu I and Kpn I sites map up-

stream of the coding sequence of the folC gene, as expected (Figure 1).

These sites are not located in a non coding region

but within the coding sequence of an upstream open reading frame. This open reading frame is 912 base pairs long and predicts a protein with a molecular weight of 33,000 daltons.

The non-coding

735 region between the two genes is only 72 bp in length.

Deletion of

sequences upstream of the above restriction sites removes most of the upstream gene and causes a concomitant loss of amplified FPGS expression in transformants.

This suggests that the promoter

function being deleted from the folC gene is the promoter of the upstream gene and that the two genes are co-regulated at the level of transcription.

I

2

3

Figure 1. Physical map of the region of the folC gene showing the open reading frames for the folC gene and the upstream gene. SDS polyacrylamide gel electrophoresis was performed to determine whether the levels of FPGS activity can be correlated with the intensity of the protein bands for the products of the folC and upstream genes.

A protein band which comigrates with purified FPGS

can be seen in extracts of amplified strains, such as transformants of pAC3, but not of wild type or transformants of plasmids with the upstream gene deleted enzyme activity.

(pAC5), which do not have amplified

Initially, we were unable to see a similar in-

crease in intensity of a band corresponding to the predicted size of the upstream gene product in these soluble extracts.

However,

when proteins in the sonic pellets were run on SDS gels, a protein band of Mr 33,000 can be seen which is more intense in extracts from transformants of pAC3 but not of pAC5 or in wild type.

Discussion We have shown that deletion of an upstream gene results in greatly decreased expression of FPGS from the downstream folC gene.

This

suggests that the promoter function responsible for high expression of the folC gene is the promoter of the upstream gene and that the

736 two gene products may be expressed from a polycistronic message. Plasmids with the upstream gene deleted express low but measurable amounts of FPGS.

They are able to complement the SF4 mutation,

suggesting that some wild type enzyme is produced.

This is true

even in plasmids, such as pAC5, in which all promoters present in the vector are oriented in the direction opposite to folC transcription.

This suggests that there may be a weak promoter in the

non coding region between the two genes.

There are sequences in

this region which have homology with the consensus sequences for the -35 and -10 promoter regions.

Studies to determine whether

there is a promoter in this region are currently in progress. We have shown that the upstream gene codes for a protein product and that its expression is coregulated with that of FPGS. function of upstream gene product is unknown at present.

The Its se-

quence has no homology with any gene whose sequence has been previously studied.

It would be very interesting to determine if the

function of this gene product is related to folate metabolism.

Acknowledgement This research was supported by grant no. CA 41991 from the National Cancer Institute, Department of Health and Human Services.

References 1.

Bognar, A.L., C. Osborne, B. Shane, S.C. Singer, R. Ferone. 1985. J. Biol. Chem. 260, 5625.

2.

Ferone, R., S.C. Singer, M.H. Hanlon, S. Roland. 1983. Chemistry and Biology of Pteridines (Blair, J.A., ed.) de Gruyter, Berlin, p. 585.

In:

O C C U R R E N C E A N D S Y N T H E S I S OF a - G L U T A M A T E S IN E S C H E R I C H I A

R. F e r o n e , M .

Hanlon,

Burroughs Wellcome

D.

S.

PTEROYL-y-GLUTAMYL-y-GLUTAMYL-POLYCOLI

Singer

Company,

Research

Triangle

Park, N.C.

27709

Hunt

Department VA 22901

of C h e m i s t r y ,

University

of V i r g i n i a ,

Charlottesville,

Introduction

In m o s t mate the

cells

analysed,

conjugates,

folate

in w h i c h

cofactors

the amide

y-carboxyls

of

the glutamates

as the p o s i t i o n

of

linkage originally

only

synthetic

L. c a s e i

assays

factor")

(1).

for

PteGlu^

a-COOH

linkages

the p r e s e n c e

to p o l y g l u t a m a t e

Results

been

in t h e

chains

y-COOH bond

impure.

We

bonds was

report

Discussion

E. c o l i w a s

grown with

[7-

14

group

to be

here

y-COOH that

as a c t i v e

in

("L.

casei

have been

the

via

finding

susceptibility

although

folylpolyglutamates

a-COOH

polygluta-

of t h e

from t h e

linkages

of t h e i r

conjugases,

via

as

Assignment came

assumed

to

cleav-

preparations

the p r e s e n c e

extracted

in E. c o l i of an e n z y m e w h i c h

and

and

found

are presumed

pteroyltriglutamate

because

p r e p a r a t i o n s of

used have usually

and

then,

folylpolyglutamates

age w i t h

(1).

containing y-COOH

as an e x t r a c t e d Since

are

bonds

adds

of

from E.

linkages.

C]p-aminobenzoate

to

label

folates

then extracted

F o o et al.

(2).

and c l e a v e d to p A B G l u . . , as d e s c r i b e d by (n) 14 The C - l a b e l l e d p A B G l u , . from E. c o l i d i d n o t (n) —

co-chromatograph

with

systems

and

Control

experiments

materials

coli

glutamates

on a Biogel

effected

synthetic

P-4 column

eliminated the

pAB(y)Glu^_^

cleavage

(as a z o - d y e

the p o s s i b i l i t y or H P L C of

on

several

HPLC

derivatives). that

E.

the p A B G l u ,

Chemistry and Biology of Pteridines 1986 © 1986 Walter d e Gruyter & Co., Berlin • New York - Printed in Germany

coli the

738 cleavage and HPLC behavior of standards (y-COOH linked) were not changed when "spiked" into E. coli extracts. The pABGlu^j derived from the cleavage of E. coli folylpolyglutamates were purified by HPLC on C, 0 Nova-pak columns eluted with acetonitrile/ 1o trifluoroacetic acid. Molecular and fragment ions of methylated unknowns, as determined by tandem mass spectrometry, were consistent with the structure of a linear chain of glutamates attached to a p-aminobenzoyl group.

Chiral HPLC on a B-cycl-odex-

trin column of dansylated glutamates formed from hydrolysed E. coli-derived p A B G l u ^

showed the glutamates to be exclusively

the L-enantiomer. The y-COOH linked pABGlu^j standards and the E. coli-derived pABGlu^ n j differed in their susceptibility to digestion by the a-COOH specific enzyme, carboxypeptidase Y.

This enzyme was shown

to cleave authentic pAB-y -Glu-a-Glu., to pAB-y-Glu.,, but not to react with pAB(y)Glu 2 _ 7 .

Purified E. coli pABGlu 4 _ g were each

digested by carboxypeptidase Y to pAB(y)Glu^, in a stepwise manner.

E. coli pABGlu^ was not digested, and co-chromatographed

with pAB (y ) Glu^ .

Thus, in E. coli folylpolyglutamates, glutamates

added after the third residue are linked via a-COOH groups (see Figure 1). FIGURE 1 y-COOH LINKED FOLYLPOLYGLUTAMATES

COOH .¿H

PTERIN—CHjNIR)-^0^-CONH 1)

pABGlu. r Glu

(n>l)

(n> 1)

aciug a-COOH linkage

740 References 1.

Krumdieck., C.L., Tamura, T., Eto, I. 1983. In: Vitamins and Hormones. Vol. 40, Academic Press, Inc., New York, p. 45.

2.

Foo, S.K., Cichowicz, D.J., Shane, B. 1980. 107, 109.

3.

Bognar, A.L., Osborned, C., Shane, B., Singer, S.C., Ferone, R. 1985. J. Biol. Chem. 260, 5625.

Anal. Biochem.

SUMMARY FOLYLPOLYGLUTAMATE SYNTHESIS Edwin A. Cossins Department of Botany, University of Alberta, Canada, T6G 2E9

Since the Symposia held

in La Jolla

(1978) and St. Andrews (1982) the

subject of folylpolyglutamate synthesis has been actively pursued of

in a number

In this regard, there has been noteable progress in studies

laboratories.

of folylpolyglutamate therefore concentrated

synthetase

(FPGS).

on recent and

The presentations

in Session 14

innovative work on this key enzyme of

folate metabolism. The 'state of the art' lecture was delivered the

general

characteristics

by Barry Shane who reviewed

of the bacterial,

fungal and manmalian

synthetases. The complete purification of the bacterial and porcine liver enzymes has allowed detailed study of the physical and catalytic properties of these proteins.

More recently, Shane's group has

sequenced the FPGS gene of E. coli. physiological

role of FPGS

suggested that cellular

Dr. Shane also considered

in the generation of native

folates. It was

Folylpolyglutamate

synthesis

affinities of FPGS for folate

in manmalian tissues

process and FPGS displays a decreased activity with substrates.

the

folate pools and the distribution of polyglutamates

tend to reflect the substrate specificities and substrates.

successfully cloned and

is a slow

longer chain polyglutamate

The lecture also included new data on the inhibition of FPGS by

pteryol-ornithine

derivatives. Replacement of glutamate by ornithine in folate

or antifolates resulted in marked inhibition of FPGS activity. Inhibition of human cell FPGS activity was the subject of the paper by J.J. McGuire and J.R. Piper. Compounds of special interest in this work were folate and antifolate analogs in which glutamate was diaminoalkanoic

acids.

replaced by various

2, omega-

Enzyme activity was strongly inhibited as the analog

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

742

side chain was lengthened to the pentanoate derivative. These compounds were however less cytotoxic to human leukemia cells than methotrexate. data presented

Fran the

it is clear that these compounds have potential as specific

inhibitors of polyglutamate synthesis. The paper by Bognar et al reported FPGS activity.

Methods were described for cloning the fol C

determining its sequence.

An open reading

upstream of the FPGS gene protein.

new work on the expression of 12. coli gene and for

frame of 910 base pairs occurs

and this region encodes for a membrane-associated

Evidence was presented that the expression of both genes was

coregulated. The

last paper

of Session 14 was

given by Dr. R. Ferone.

In an

investigation with Hunt, Hanlon and Singer the polyglutamates of E. coli were shown to contain glutamyl noieties, linked via first three glutamates

are

linked by

a- and y-carboxyl bonds. The

Y-carboxyl bonds whereas additional

glutamates are linked via their a-carboxyl groups.

Cell-freee

extracts of E.

coli were shown to contain an enzyme that produced such a-carboxyl products in vitro.

This activity was separated frcm FPGS-dihydrofolate synthetase by gel

filtration. It was concluded

that

folylpolyglutamate formation in E. coli

involved two distinct glutamyl conjugation reactions.

THE METABOLISM OF PTEROYLPOLYGLUTAMATES

Roy L. Kisliuk Department of Biochemistry and Pharmacology, Tufts University, Boston, Massachusetts 02111

Introduction The most thoroughly documented early study suggesting the existence of polyglutamyl derivatives of folate was that of Ratner, Blanchard and Green (1) who, in 1946, described the isolation of a p-aminobenzoic acid derivative from yeast which contained 10-11 glutamic acid residues. These authors included an addendum which states "Some time after this manuscript was submitted for publication, the structure of folic acid was published (2). It appears that an essential part of the folic acid molecule is analogous to the PAB peptide described here. There remains to be determined, therefore, whether a functional relationship exists between the PAB peptide of yeast and the various conjugates of folic acid." That such a functional relationship does indeed exist has been amply documented and reviewed (3-10). We here briefly review selected areas of folate polyglutamate metabolism under active study including: intestinal absorption, biosynthesis, biodégradation, metabolic chain length alterations, interactions with folate enzymes and enzymeenzyme interactions. We also discuss methanopterin, an unusual folate-like coenzyme involved in methane formation. Intestinal Absorption Decisive studies in this area were made possible by the development of the solid phase synthesis of model folate polyglutamates (11). Intestinal absorption depends on the hydrolysis of dietary polyglutamates which takes place at the mucosal border (12-14). A zinc-requiring exopeptidase has been isolated from the brush border membrane of human intestinal cells (15). This enzyme differs

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

744 from an intracellular hydrolase isolated from the same tissue. Although mucosal cells can convert folate to 5-methyltetrahydrofolate, the main blood form of folate, reduction, methylation and presumably polyglutamylation are not required to transport folate across the villus tip cells (16,17). It is usually assumed that dietary folates are poly-gamma-glutamyl forms but it has recently been reported that E. coli contains folate polyglutamates in which the first three Glu residues are linked through their gamma-carboxyl groups but additional Glu residues are alpha-linked (18) . In addition, the enzyme folate oligoglutamate transpeptidase has been described in rat liver extracts which catalyses the exchange of the terminal Glu residue with methionine, glutamine or glycine (19). Perhaps further studies will reveal that dietary folates contain unusual peptide chains. Biosynthesis Most biological systems convert folates to polyglutamate forms (10). The action of a single enzyme, folylpolyglutamate synthetase, is thought to account for this synthesis in most instances. However, in Neurospora crassa at least two enzymes are involved (20) . One converts H 4 PteGlu 1 to H 4 PteGlu 2 and a second enzyme converts H.PteGlu_ to H.PteGlu,. A third activity found in mitochondria, 4 Z 4 b catalyses the conversion of H 4 PteGlu 2 to H4PteGlu3- E. coli contains two enzymes for folylpolyglutamate synthesis. One is the combined folylpolyglutamate synthetase-dihydropteroate synthetase which can catalyse the synthesis of derivatives containing three gamma-linked Glu residues. The gene for this enzyme has been cloned (21). A second enzyme catalyses the further addition of alpha-linked Glu residues (181• The substrate specificities for many folylpolyglutamate synthetases have been summarized (5,6,10). More recently studies with hog liver (22), mouse liver (23), beef liver (24) and human liver (25) have been carried out. These enzymes all have a broad specificity with respect to the pteridine moiety of folates and folate analogs but they require a free amide carboxyl group on the Glu residue of the folate or antifolate for activity. 5-Methyltetrahydrofolate is generally a poor substrate. The length of the polyglutamate chain synthesized often depends on the substrate offered.

745 DL-threo-4-Fluoroglutamic acid is a chain terminating inhibitor of rat liver folylpolyglutamate synthetase (26) . A methotrexate derivative containing fluorglutamic acid proved to be a potent inhibitor of dihydrofolate reductase but was not converted to polyglutamate forms by H35 hepatoma cells and was considerably less toxic than methotrexate (27) . These results suggest that polyglutamylation is an important component of the cytotoxic effect of methotrexate. Biodégradation It is usually assumed that gamma-Glu-hydrolases commonly found in animal tissues are involved in the biodégradation of folylpolyglutamates but this has not been definitively established (10). In most instances activity is located in the lysosomes (28) . Specificity for folylpolyglutamates is not generally observed (10) but the intracellular hydrolase from human intestine requires the folate portion of the molecule (15). Recent studies on the hydrolysis of methotrexate polyglutamates in murine tissues show that activity is much lower in extracts of ascites tumors (Sarcoma 180, Ehrlich, L1210) than in extracts of liver, small intestine, bone marrow or kidney (29). The tumor cells showed highest activity when harvested during the early logarithmic phase of growth. Activity of the tumor cells grown in vitro was lower than activity found in the ascites forms. A striking instance of a metabolic role for a gamma-Glu-hydrolase is suggested by the studies on the formation of F^PteGlUg from longer chain polyglutamates in E. coli infected with T4 bacteriophage (30). H2PteGlUg is specifically needed as a structural component of the baseplate of this bacteriophage as is the gamma-Glu-hydrolase itself. Alterations in Folylpolyglutamates During Metabolism Studies on the alterations in the chain length of folylpolyglutamates under different dietary or growth conditions were recently reviewed (8). There is a shift to longer chains in Neurospora grown on a high glycine medium (31) , in Lactobacillus casei grown on a low folate medium (32), in quail liver from animals maintained

746 on a high casein diet (33) , and in rats maintained on a folate deficient diet (34). In cultured mouse hepatoma cells starved for folate for 48 hours, GlUg is the predominant folate polyglutamate as opposed to the normal distribution of equal amounts of Glu^ and GlUg (35). These shifts to longer chain lengths may bring about more efficient use of limited amounts of folate. During rat liver regeneration, GlUg and Glu^ derivatives increase at the expense of Glu^ and Glu,. forms (36) . The newly synthesized GlUg forms are predominantly 5-methyltetrahydrofolate, whereas the newly synthesized Glu^ forms are formylated derivatives. Other studies on rat liver (37) found that different H^PteGlu predominate in fractions of rat liver separated according to chain length. Among Glu,. derivatives, 5-methyltetrahydrofolate is the predominant form whereas the Glu^ derivatives are mainly H^PteGlu. These results suggest that derivatives containing different chain lengths may serve different functions and may be located in different cellular compartments. Mouse Sarcoma 18 0 cells have larger folate pools with longer Glu chains than do human Hep-2 carcinoma cells (38) . This observation provides a reasonable explanation for the greater susceptibilty of the mouse cells to 5-fluorouracil, which after conversion to 5fluorodeoxyuridylate, binds to thymidylate synthase more firmly in the presence of methylenetetrahydrofolate derivatives. Improved methods of analysis for tissue folates (39,40) and polyglutamate chain length (41,42,43)have yielded new information on the effects of inhibitors on folate metabolism. For example, methotrexate treatment leads to a decrease in the levels of 5-methyltetrahydrofolate in L1210 cells (43) and MCF-7 cells (40) . This apaprently results from accumulation of dihydrofolate polyglutamates which inhibit the reduction of methylenetetrahydrofolate. Interaction of Folylpoyglutamates with Enzymes A. Regulatory interactions: Polyglutamyl derivatives of 5-methyltetrahydrofolate are known to regulate the activity of at least three folate enzymes; cystathionine-gamma-synthase of Neurospora (44), serine hydroxymethyltransferase of pig liver (45) and glycine N-methyl transferase from rat liver (46). Cystathionine-gamma-synthase, which catalyses a reaction on the

747

on the biosynthetic pathway to homocysteine, requires 5-methyltetrahydrofolate as an allosteric activator. The Glu 7 form is much more potent than the Glu^ form. Thus the methylated coenzyme stimulates the formation of the acceptor of its methyl group and methionine is formed. The weak inhibition of serine hydroxymethyltransferase by 5-methyltetrahydrofolate (47) is enhanced by the elongation of the Glu chain. Inhibition of this enzyme leads to a decrease in the amount of serine used to supply single carbon units for purine, thymine and methionine formation and an increase in the amount of serine available for gluconeogenesis or oxidative metabolism. Glycine N-methyl transferase is a major folate binding protein in rat liver (48). It catalyses the reaction: glycine plus S-adenosylmethionine yields sarcosine plus adenosylhomocysteine. This reaction does not require a folate coenzyme, but is strongly inhibited by the Glu,- derivative of 5-methyltetrahydrofolate (46) . The glycine N-methyltransferase reaction provides a mechanism for removing excess adenosylmethionine which might arise from dietary methionine. Both products of the reaction can be converted to useful metabolites. Adenosylhomocysteine can be split and rearranged to form methionine (49), whereas sarcosine is converted to glycine and methylenetetrahydrofolate in mitochondria (50). B: Coenzyme Interactions: Only one folate enzyme is known to have a stringent requirement for a folylpolyglutamate coenzyme, the nonB^2 requiring 5-methyltetrahydrofolate-homocysteine transmethylase from E. coli (51) or B. subtiltis (52). Other folate enzymes will function with Glu^ derivatives although addition of gamma-Glu residues generally enhances affinity of cofactors for folate enzymes (8-10). One likely role for the polyglutamate chain is to aid in the channeling of folate cofactors among sequential folate enzymes. A role for folylpolyglutamate substrates in substrate channeling has been demonstrated for the two reactions involved in the conversion of formiminoglutamic acid to methenyltetrahydrofolate catalysed by fromiminoglutamate:tetrahydrofolate formiminotransferase and formiminotetrahydrofolate cyclodeaminase from pig liver (53). These enzymes are associated as a tetramer of dimers which binds four pteroylpolyglutamates per octamer. The efficiency of channeling was highest with the pentaglutamate although binding was

748

tightest with the hexaglutamate. This demonstrates that channeling has a steric requirement which can be separated from the affinity of the system for the ligand. Enzyme-Enzyme Interactions Evidence is accumulating which shows that the common conditions used for enzyme assay, in which the enzyme concentration is low relative to the substrate concentration, are a poor reflection of the situation in vivo where the situation is reversed. An example is phosphofructokinase (54) where the widely cited (55) inhibition by ATP is greatly decreased when the enzyme is studied 1) at high concentration (0.6 mg/ml), 2) in permeabilized red blood cells or 3) in the presence of polyethylene glycol which increase the local protein concentration. The authors go so far as to state that "... most kinetic data obtained with routinely diluted enzyme are not extrapolable for quantitatively meaningful regulation in vivo." This is a sobering thought for those of us who always carry out assays with dilute enzyme. High enzyme concentration favors channeling, that is the transfer of ligands from enzyme to enzyme without dispersion in the solvent. An interesting example of this type of transfer occurs between NADH and NADH linked dehydrogenases (56,57). NADH can pass directly from glyceraldehyde-3-phosphate dehydrogenase to alcohol dehydrogenase. Direct transfer occurs when the two enzymes transfer hydrogen from opposite faces (A and B) of the nicotinamide ring. Whenever the two enzymes involved are both A specific or both B specific, transfer occurs through the solvent. Consideration of the molecular basis for the transfer of NADH from one dehydrogenase to another based on the crystal structures of glyceraldehyde-3-phosphate dehydrogenase"and liver alcohol dehydrogenase enables estimation of the electrostatic potential of the enzyme surfaces likely to be involved in docking (58). The A specific dehydrogenases examined have areas of negative potential around the opening designed for the nicotimamide portion of the coenzyme. It is postulated that after the nicotinamide portion of the coenzyme transfers from one enzyme to the next, the remainder of the molecule rotates 180° at the Nj-C^' glycosidic bond before binding to the acceptor enzyme. It is noteworthy that a similar

749

180° rotation is possible in the binding site of dihydrofolate reductase in that the pteridine ring of methotrexate is rotated 180° from that of dihydrofolate about the C g - N 1 0 bond. Future studies of the relationship of folate polyglutamates with their corresponding enzymes will have to deal with the interactions between sequential enzymes as in the thoroughly documented case of the channeling interaction between formiminotransferase and cyclodeaminase mentioned above. We believe a fruitful area for such studies is to be found in the thymidylate cycle. In the animal and bacterial systems so far studied, the three enzymes of this cycle, dihydrofolate reductase, serine hydroxymethyltransferase and thymidylate synthase have not been shown to associate physically. In protozoa, however, dihydrofolate reductase and thymidylate synthase share the same peptide chain (59). The thymidylate cycle has been studied extensively because dihydrofolate reductase and thymidylate synthase are the target enzymes for widely used chemotherapeutic agents. Our attention was particularly drawn to a paper by Harvey (60), who gives a well- reasoned explanation of the antibacterial synergy observed when sulfonamides, which inhibit dihydrofolate synthesis, are combined with trimethoprim, a potent inhibitor of bacterial dihdyrofolate reductase. The essence of the argument is that when trimethoprim is present alone, it will inhibit dihydrofolate reductase until enough dihydrofolate (polyglutamate), generated by the action of thymidylate synthase, accumulates to compete effectively with trimethoprim and overwhelm the block. Reduction of the level of folate coenzyme in the cycle through the action of sulfonamides on folate biosynthesis would render the system more sensitive to trimethoprim because the amount of competing dihydrofolate coenzyme would be lowered. If two enzymes within the cycle are inhibited, antagonism is predicted because only one of the enzymes within the cycle could be rate limiting. Therefore inhibiting the other would be ineffective (61) . We felt that the interaction of the three enzymes in vivo might be more complicated so we tested the growth inhibtion of L. casei brought about by combinations of trimethoprim to inhibit dihydrofolate reductase and 5,8-dideaza-10-propargylPteGlu, a powerful inhibitor of thymidylate synthase (62-64)under conditions where thymidylate is the growth limiting metabolite. Synergistic growth inhibition

750

was obtained with this combination (65). With the Glu 2 derivative of the propargyl derivative, antagonistic, additive or synergistic effects were obtained with increasing concentration. In order to eliminate the possibility of the inhibition being influenced by the transport or metabolism of folates or inhibitors, a system was developed consisting of two enzymes of the cycle, dihydrofolate reductase and thymidylate synthase purified from extracts of L. casei (66). Formaldehyde is the source of the single carbon unit and net thymidylate synthesis is measured under steady state conditions by release of H from 5-(^H)-dUMP. Table 1 Inhibition of Thymidylate Formation by Combinations of Trimethoprim with 5,8-Dideaza-10-propargylPteGlu Derivatives Expt. 1 Cone (M) 1.6 3.3 6.6

X

X

10" 10 10" 10 10" 10

13.2

X

10" 10

X

2 1.7 3.4 6.8 13.6

PPG1 alone

TMP alone

TMP + PPG^

% Inhib.

% Inhib.

% Inhib. Cale. Obs.

1 11 21 36 PPG 2 alone

X X X X

10" 11 11

10" 10" 11 10"

11

0 4 18 34

22 22 22

23 33 43 58

22 TMP alone 10 10 10 10

*

23 24 35 48

TMP + PPG 2 10 14 28 44

14 19 19 36

Abbreviations: PPG-j^ = 5,8-dideaza-lO-propargylPteGlu-j^; PPG 2 5,8-dideaza-10-propargylPteGlu2; TMP = trimethoprim Conditions described in (66) . * TMP conc. 5 x 10 8 M. Folate cofactor added

=

H 2 PteGlu 1 at 1.3 x 10 5 M, NADPH 5 x 10

5

M

Table 1 shows data comparing the inhibition obtained with combinations of trimethoprim with PPGj^ and PPG2- With PPG 1 (Expt. 1) the combination results in antagonism wheras with PPG 2 at lower concentrations a small but reproducible synergistic effect is obtained. The concentration of PPG 2 at which this effect can be observed, 1-3 x 1 0 - 1 1 M, is about 1/100 that of the molar concen-

751 of thymidylate synthase present. The ratio of activities of dihyrofolate reductase to thymidylate synthase in the system is 4:1. The mechanism of the synergistic effect is not known but it may relate to the steady state concentrations of dihydrofolate and tetrahydrofolate. Under conditions where TMP is inhibiting1dihydrofolate reductase, the level of dihydrofolate piling up behind the block would be reduced by inhibition of thymidylate synthase. As inhibition of thymidylate synthase is increased, it becomes rate limiting and the block of dihydrofolate reductase becomes less influential. In this in vitro system, synergism is obtained at low PPG2 concentrations and antagonism at higher concentrations which is opposite to the results obtained in vivo (65). This in vitro system will allow us to study effects of polyglutamate cofactors and inhibitors as well as to compare formaldehyde versus serine plus serine hydroxymethyltransferase as sources of methylenetetrahydrofolate derivatives. It should also enable us to study potential direct interactions between the three enzymes of the cycle. Methanopterin Methanopterin derivatives which are coenzymes of one-carbon metabolism in methane bacteria (67) are not known to occur as polyglutamates. However their structure is truly unique (Figure 1) and the methods used in studies of their structure and biosynthesis aré "state of the art". In addition, they may be considered as analogs of folate polyglutamates in which the Glu chain is replaced by the two pentose units and a phosphate in diester linkage with alpha-hydroxyglutaric acid.

OH

Figure 1

Tetrahydromethanopterin (71)

COOH

752 The structure of methanopterin was elucidated using two-dimensional NMR techniques (68) , whereas that of methenyltetrahydromethanopterin confirmed the results of NMR studies with fast atom bombardment mass spectrometry (69) . The biosynthetic pathway has been stu13 14 died by the incorporation of C (70) and C (71) compoundsi Methanobacterium thermoautotrophicum, from which the methanopterin derivatives were isolated, has less than 1% the level of folate found in E. coli or B. subtilis (72). This indicates that onecarbon metabolism in this organism utilizes methanopterin cofactors in place of folates. We thought it would be of interest to test methanopterin derivatives in some standard folate test systems. Dr. G.D. Vogels of the University of Nijmegen kindly provided us with a sample of methanopterin from which we prepared the corresponding dihydro and tetrahydro derivatives. No growth promoting activity was seen for Streptococcus faecium (ATCC 8043) or Lactobacillus casei (ATCC 7469) at 100 nanograms/ml with any of the three compounds. Folate gives full growth at 1 nanogram/ml. No growth inhibition of these organisms was seen with these three compounds at 1 microgram/ml. In addition, dihydromethanopterin was neither a substrate -5 nor an M. inhibitor of L. casei dihdyrofolate reductase at 5 x 10 Tetrahydromethanopterin was not a substrate for L. casei thymidy-4 late synthase at 3 x 10 M even at 10 times the usual enzyme concentration. These results emphasize the unique character of the methane bacteria since folate derivatives are ubiquitous in other life forms as far as is known. (It should also be mentioned that a gamma-linked diglutamate derivative of 8-hydroxy, 7-demethyl, 5-deazaflavin has been isolated from methane bacteria (67).) Acknowledgement Work in the authors laboratory was supported by Grant CA 10914 from the National Cancer Institute and carried out with outstanding skill by Ms Yvette Gaumont and Dr. Henry Rebandel.

753 References 1. Ratner, S., M. Blanchard, D.E. Green. 1946. J. Biol. Chem. 164, 691. 2. Angier, R.B., J.H. Boothe, B.L. Hutchings, J.H. Mowat, J. Semb, E.L.R. Stokstad, Y. SubbaRow, C.W. Waller, D.B. Cosulich, M.J. Fahrenbach, M.E. Hultquist, E. Kuh, E. H. Northey, D.R. Seeger, J.P. Sickels, J. M. Smith, Jr. 1946. Science 103, 667. 3. Stokstad, E.L.R., J. Koch. 1967. Physiol. Rev. £7, 83. 4. Baugh, C.M. , C.L. Krumdieck. 1971 Ann. N.Y." Acad. Sei. 186, 7. 5. Mc Guire, J.J., J. R. Bertino. 1981. Mol. Cell. Biochem. 3£, 19. 6. Cichowicz, D.J., S.K. Foo, B. Shane. 1981. Mol. Cell. Biochem. 39., 209. 7. Kisliuk, R.L. 1981. Mol. Cell. Biochem. ¿9, 331. 8. Kisliuk, R.L. 1984. In: Folate Antagonists as Therapeutic Agents (F.M. Sirotnak, J.J. Burchall, W.D. Ensminger and J.A. Montgomery, eds.) Academic Press, p. 1. Vol. 1. 9. Goldman, I.D. (ed.). 1985 Proceedings of the Second Workshop on Folyl and Antifolyl Polyglutamates, Praeger. 10. McGuire, J.J., J.K. Coward. 1984. In Folates and Pterins, Vol. 1. (R.L. Blakley and S.J. Benkovic, eds.) Wiley, p. 136. 11. Krumdieck, C.L., C.M. Baugh. 1969. Biochemistry 8, 1568. 12. Halsted, C.H. 1979. Am. J. Clin. Nutr. 32,

846.

13. Selhub, J., G.J. Dhar, I.H. Rosenberg. 198 3. Pharmacol. Ther. 20, 397. 14. Elsenhans, B., 0. Ahmad, I.H. Rosenberg. 1984. J. Biol. Chem. 259, 6364. 15. Wang, T.T.Y., C.J. Chandler, C.H. Halsted. 1986. Fed. Proc. £5, 479. 16. Selhub, J., H. Brin, N. Grossowicz. 1973. Eur J. Biochem. 3J3, 433. 17. Selhub, J., G.M. Powell, I.H. Rosenberg. 1984. Am J. Physiol. 246,G515. 18. Hanion, M.R., R. Ferone, S. Singer, D. Hunt. 1986. Fed. Proc. £5, 1542. 19. Brody, T., E.L.R. Stokstad. 1982. J. Bio-. Chem. 257, 14271. 20. Cossins, E.A., P.Y. Chan. 1984. Phytochemistry 23, 965.

754 21. Bognar, A.L., C. Osborne, B. Shane, S.C. Singer, R. Ferone. 1985. J. Biol. Chem. 260, 5625. 22. Cichowicz, D., J. Cook, S. George, B. Shane. 1985. In: Second Workshop on Folyl and Antifolyl Polyglutamates, (I.D. Goldman, ed.) Praeger, p.7. 23. Moran, R.G., P.D. Coleman, A. Rosowsky, R.A. Forsch, K.K. Chan. Mol. Pharmacol. 156. 24. Schoo, M.M.J., Z.B. Pristupa, P.J. Vickers, K.G.Scrimgeour. 1985. Cancer Research. 45, 3034. 25.

Waxman, D.J., L. Clarke. 1986. Proc. Am Assoc Cancer Res. 27, 255.

26. McGuire, J.J., J.K. Coward. 1985. J. Biol. Chem. 260,6747. 27. Galivan, J., J. Inglese, J.J. McGuire, Z. Nimec, J.K. Coward. 1985. Proc. Natl. Acad Sei. USA 82^, 2598. 28. Priest, D.G., C.D. Veronee, M. Mangum, J.M. Bednarek, M.T. Doig. 1982. Mol. Cell. Biochem. 4_3, 81. 29. Samuels, L.L., Goutas, L.J. D.G. Priest, J.R. Piper, F.M. Sirotnak. 1986. Cancer Research £6, 2230. 30. Kozloff, L.M. 1985. In: Folyl and Antifolyl Polyglutamates (I.D. Goldman, ed. ) Praeger, p. 22. 31. Chan, P.Y., E.A. Cossins. 1980. Arch. Biochem. Biophys. 200, 346. 32. Shane, B., A.L. Bognar, R.D. Goldfarb, J.H. LeBowitz. 1983. J. Bact. 153,316. 33. Thompson, R.W., J. Leichter, P.E. Cornwall, C.L. Krumdieck. 1977. Am. J. Clin. Nutr. 30, 1583. 34. Cassady, I.A., M.M. Budge, M.J. Healy, P.F. Nixon. 1980. Biochem Biophys Acta. 633, 258. 35. Priest, D.G., Doig, M., Mangum, M. 1983. Biochem. Biophys. Acta. 756, 253. 36. Eto, I., C.L. Krumdieck. 1982. Life Sciences 30, 183. 37. Brody, T., J.E. Watson, E.L.R. Stokstad. 1982. Biochemistry 21, 276. 38. Yin, M.B., S.F. Zakrzewsky, M.T. Hakala. 1983. Mol Pharmacol. 23, 190. 39. Wilson, S.D., D.W. H o m e . 1986. Arch. Biochem. Biophys. 244, 248. 40. Allegra, C.J., R.L. Fine, J.C. Drake, B. Chabner. 1986. J. Biol. Chem. 261, 6478.

755

41. Eto, I., C.L. Krumdieck. 1981. Anal. Biochem. 115, 133. 42. Priest, D.G., K.K. Happel, M. Mangum, J. M. Bednarek, M.T. Doig, C.M. Baugh. 1981. Anal. Biochem. 115, 163. 43. Doig, M.T., J.R. Peters, P. Sur, M. Dang, D.G. Priest. 1985. J. Biochem. Biophys. Met. H), 287. 44. Selhub, J., W. Sakami, M. Flavin. 1971. Proc. Natl. Acad. Sci. USA 312. 45. Matthews, R.G., J. Ross, C.M. Baugh, J.D. Cook, L. Davis. 1982. Biochemistry 21,1230. 46. Wagner, C., W.T. Briggs, R.J. Cook. 1985. Biochem. Biophys. Research. Commun. 127, 746. 47. Schirch, L., M. Ropp. 1967. Biochemistry 6, 253. 48. Cook, R.J., C Wagner. 1984. Proc. Natl. Acad. Sci. USA. 81, 3631. 49. Backlund, P.S. Jr., C.P. Chang, R.A. Smith. 1982. J. Biol. Chem. 257, 4196. 50. Wittwer, A.J., C. Wagner. 1981. J. Biol. Chem. 256, 4109. 51. Guest, J.R., S. Friedman, M.A. Foster, D.D. Woods. 1964. Biochem. J. 92^, 497. 52. Salem, A.R., J.R. Patterson, M.A. Foster. 1972. Biochem. J. 126, 993. 53. Pacquin, J. C.M. Baugh, R.E. Mackenzie. 1985. J. Biol. Chem. 260,14925. 54. Bosca, L., J.J. Aragon, A. Sols. 1985. J. Biol. Chem. 260, 2100. 55. Orten, J.M., Neuhaus, O.W. 1982. Human Biochemistry, C.V. Mosby, p. 229. 56. Srivastava, D.K., S.A. Bernhard. 1985. Biochemistry, 24, 623. 57. Srivastava, D.K., S.A. Bernhard. 1986. In: Current Topics in Cellular Regulation and Metabolism (B.L. Horecker and E.R. Stadtman, eds.) Academic Press (in press). 58. Srivastava, D.K., S.A. Bernhard, R. Langridge, J.A. McLarin. 1985. Biochemistry, 24, 629. 59. Ferone, R., S. Roland. 1980. Proc. Natl. Acad. Sci. USA 77, 5802. 60. Harvey, R.J. 1982. Rev Inf. Diseases, 4, 255. 61. Jackson, R.C., A.L. Jackman, A.H. Calvert. 1983. Biochem.

756 Pharmacol. 32^ 3783. 62. Jones, T.R., A.H. Calvert, A.L. Jackman, S.J. Brown, M. Jones, K.P. Harrap. 1981. Eur. J. Cancer, 17, 11. 63. Nair, M.G., D.C. Salter, R.L. Kisliuk, Y. Gaumont, G. North, F.M. Sirotnak. 1983. J. Med. Chem. 26, 605. 64. Nair, M.G. , N.T. Nanavati, I.G. Nair, R.L. Kisliuk, Y. Gaumont, M.C. Hsiao, T. Kaiman. J. Med. Chem. (in press). 65. Kisliuk, R.L., Y. Gaumont, P. Kumar, M. Coutts, M.G. Nair, N.T. Nanavati, T.I. Kaiman. 198 5. In: Second Workshop on Folyl and Antifolyl Pölyglutamates,(I.D. Goldman, ed.) Praeger, p.319. 66. Rebandel, H., Gaumont, Y., R. L. Kisliuk. (This volume). 67. Wolfe, R.S., Trends In Biocemical Sciences. 1985. 10,396. 68. Van Beelen, P., A.P.M. Stassen, J.W.G. Bosch, G.D. Vogels, W. Guijt, C.A.G. Haasnoot. 1984. Eur. J. Biochem. 138, 563. 69. Van Beelen, P., J.W. Van Neck, R.M. deCock, G.D. Vogels, W. Guijt, C.A.G. Haasnoot. 1984. Biochemistry 23^, 4448. 70. Keller, P.J., H.G. Floss, Q. Le Van, B. Schwarzkopf, A. Bacher. 1986. J. Am. Chem. Soc. 108, 344. 71. White, R.H. 1986. J. Bacteriol. 165, 215. 72. Leigh, J.A. 1983. Applied and 800.

Environmental Microbiol. 45,

ADDENDUM This paper is dedicated to the memory of Professor Warwick Sakami (1918-1986), mentor, scientist, friend.

DIHYDROPTEROYL HEXAGLUTAMATE AND T4 PHAGE BASEPLATE ASSEMBLY

Boguslaw Szewczyk*, Krystyna Szewczyk*, and Lloyd M. Kozloff Department of Microbiology and Immunology, University of California, San Francisco San Francisco, California 94143-0404

Summary and Introduction A major step in the assembly of the T4 bacteriophage tail baseplate is the addition of six wedge-like structures around a central hub (1).

The baseplate has a mass of 7.7 xlO^ Kd, it

contains 19 different proteins of known size and six molecules of dihydropteroyl hexaglutamate

(2).

The report by Maley si. al.

(3)

that carbodiimide accivated the carboxyl groups of a labeled folyl polyglutamate and lead to the formation of covalent bonds to thymidylate synthase suggested that a similar approach could identify the baseplate proteins nearest the phage folate.

Culture

media containing l^C-aminobenzoic acid (to label only the folyl polyglutamate) was inoculated with £.. coli

and the host cells

infected with a T4 mutant, unable to form complete particles. Tail substructures containing the baseplate were purified and reacted with carbodiimide.

Six labeled protein bands were found

when tnese baseplates were analyzed by PAGE, two major bands at 41.5Kd and 39Kd, and four minor bands at 82Kd, 55Kd, 31Kd and 25Kd.

The major band at 39Kd was identified using

immunoblotting

as dihydrofolate reductase, a baseplate outer wedge component. The minor 31Kd band appeared to be phage thymidylate synthase, a baseplate hub component, since its mobility on gels was the same as the enzyme.

The other 4 labeled proteins have not been

unequivocally identified.

These results support the proposal

(1,2) that the baseplate folyl polyglutamate links the outer wedges to the baseplate hub.

*

Current address: Department of Biochemistry, University of Gdansk, 80-822 Gdansk, Poland

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

758 Materials and Methods Biological methods were similar to those used before (2).

The

major phage T4 amber mutant used was defective in genes 14 and 15 (B20/N133).

[l^c]-p-aminobenzoic acid was incorporated into the

phage T4 substructure consisting of the baseplate plus the inner tail tube as described earlier (4). dihydrofolate reductase from C.

Antiserum used were:

Mathews; anti-T4

anti-T4

thymidylate

synthase from G. Maley; anti-T4 whole baseplate from P. Berget. G. Maley also furnished purified T2 thymidylate synthase.

SDS-

PAGE, protein transfer to nitrocellulose and immunoblotting were performed by standard procedures

(6,7,8).

l-ethyl-3-(dimethyla-

minopropyl) carbodiimide hydrochloride and 4-chloro-l-naphthol were from Sigma Chemical Co., St. Louis.

Horseradish peroxidase

conjugated to goat anti-rabbit immunoglobulins was from Boehringer Mannheim.

Results and Discussion coupling

Phage Baseplate Proteins ifi.

Labeled Folate ¿¡X Carbodiimide Treatment.

-p-aminobenzoic About lOmg of the

labeled phage purified substructure containing the baseplate plus the tail tube (4) was treated with 100 mg of carbodiimide for 4 hours at 25° at pH 7.0 in 0.05 M TRIS buffer.

The usual

activation at pH 4.75 was not used because at the lower pH baseplate proteins reacted with each other forming insoluble precipitates.

Coupling at pH 7.0 did not lead to any significant

differences in SDS-PAGE profiles of untreated and carbodiimidetreated material which indicated that no measurable reaction between individual substructure proteins took place.

The proteins

of the gels of the treated labeled baseplates were transferred to nitrocellulose and subjected to autoradiography.

Densitometric

analysis revealed six weak, radioactive protein bands (Fig. 1). The two bands of highest intensity corresponded to molecular sizes of approximately 41.5 Kd and 39 Kd, and there were four additional weaker bands of 82 Kd, 55 Kd, 31 Kd and 25 Kd.

759

AL.5

K

39 k

Fig. 1. Densitometer of the radioautograph nitrocellulose of the PAGE gel of 14 C-PABA labeled T4 baseplates with carbodiimide.

tracing from SDS-

Identification fli i i

M

treated

Labeled Baseplate Protein. The direct comparison of the size of a particular gene product on a gel with that of the labeled proteins eliminated some proteins but did not alone identify the folate binding proteins since

linKing of the folate compound to a protein may have slightly altered its electrophoretic mobility (M.W. of folyl polyglutamate is - 1 Kd.

Where possible, immunoblotting was used to confirm the

identification.

When the proteins of the treated labeled

baseplates separated on gel and transferred to nitrocellulose were reacted with anti-phage T4 dihydrofolate reductase serum, only one band of 39 Kd was observed (not shown).

This band corresponded to

14

the 39 Kd protein band labeled with [ C]-p-aminobenzoic acid. The molecular weight of the monomer of phage dihydrofolate reductase (called frd) is about 20 Kd (9), hence it was possible that this anti-frd reacting band is due to the coupling of two frd monomers to the folate compound.

However, when unlabeled

baseplates, not treated with carbodiimide, were similarly immunoblotted with the anti-frd serum, a band in the same position was observed, and no band corresponding to the dihydrofolate reductase monomer was seen.

The folate binding protein of 39 Kd

is then either a modified or an unmodified dimer of two dihydrofolate reductase subunits with a covalent bond between the subunits. imrounoblotting oL Baseplate proteins With other Anti-seta.

The

molecular size of thymidylate synthase (called td) monomer is about 30 Kd.

This molecular weight corresponded to the molecular

weight of the minor protein band of 31 Kd labeled with [l4C]-paminobenzoic acid.

We could not immunologically confirm that this

760 band was td, because the anti-td serum was too weak.

However,

based on the identical mobility of this labeled band and the highly purified T2 td, and the known presence of only a few thymidylate synthase molecules in the baseplate hub, it seems likely tnat this minor labeled band is td.

Efforts were made to

identify the other labeled proteins using anti-baseplate serum and bacterial extracts obtained after infection with various phage mutants.

This baseplate anti-sera has antibodies against gene

product 12 (55 Kd) and gene product 11 (23 Kd), which were potential candidates for the minor labeled bands at 55 Kd and 24 Kd.

It was found that the smallest labeled protein could not be

the baseplate gene 11 product.

When baseplate proteins were

reacted with anti-baseplate serum, the gene 11 product band was higher than that of the labeled protein of MW 24K.

Similarly the

approximately 55 Kd band could not be gP12 since this labeled band migrated somewhat faster than gP12. Baseplate Proteins .Linked ¿s. Folyl Polyalutamate.

The identifi-

cation of the proteins linked to the labeled folate, shown in Table 1, is complicated by the possibility that the 7 activated folyl carboxyl groups plus any activated protein carboxyl groups (which could react with the amino group on the pteridine ring) could bind to more than one protein.

However, one expects that

the initial crosslinking reaction for one protein would so disturb the native structure that a second crosslinking event would become unlikely, and we have favored the view that at least the two major labeled bands represent the folate compound crosslinked to a single baseplate protein. experiments

This conclusion was supported by

(not shown) indicating that there was no non-specific

linking of the folate to basic proteins of the phage structure. The 39Kd major band is clearly the product of the phage frd gene and it is possible that the labeled folate is bound both by its amino group, to possibly the asp-27 residue

(10) of the frd and by

one of its carboxyl groups to some amino group in this protein. The other major labeled band at 41.5 Kd, which is even more highly labeled than the frd band, has not been identified.

The most

likely possibility based on size is the gene 26 product which is a hub component of 41 Kd.

The gene 26 product has not been studied

761 TABLE 1 - COMPOSITION OF THE T4D BACTERIOPHAGE BASEPLATE Molecular Size Kd

Copy Number

Total Mass Kd

Linked to Folate

77 24 49 44 41 29 16

6 -3 6 6 -3

462 72 284 264 123 58 16 1279

Likely Likely No No Likely Yes

88 24 140 46 85 23 15 20

2 4 1 1 2 1 1 -1

176 96 140 46 170 23 15 20 686

Hub 1279 Wedge 686 gP9 34 gP12 55 gP48 44 gP54 36 H 2 Pteglu 6 1

1 6 24 18 6 6 6

1279 4116 816 990 264 216 6 7687

Component Central Hub

Individual Wedge

Complete Baseplate

gP29 gP28 gP27 gP5 gP26 gPtd gP51 gPIO gPll gP7 gP8 gP6 gP53 gP25 gPfrd

~2

and its ability to bind folate is not known.

?

No No No No No No No YES -

NO No No No -

For the minor bands

we have reasonable evidence that the 31 Kd band is due to folate bound to the hub thymidylate synthase.

The band at 25 Kd probably

is due to the binding of the folate to the hub gene product 28, of 24 Kd.

This gene product is a folyl polyglutamate

carboxypeptidase

(11) and would be expected to react with

activated carboxyl groups.

The identity of the final two minor

bands of about 82 Kd and 55 Kd are less certain.

The largest

could be due to the complex of the folate plus the hub gene 29 product, of 77 Kd which is a folyl polyglutamate synthetase (12) but identity of the 55 Kd band is unknown.

These experiments show

that the dihydropteroyl hexaglutamate is near or closely associated with only 6 of the 19 baseplate proteins and clearly plays a specific structural role in this virus particle by linking together two major components.

762 Acknowledgement This investigation was supported by NIH Grant AI 18370 from the National Institute of Allergy and Infectious Disease.

References 1.

Kozloff, L.M. Dubrow, Ed.).

1981. In: Bacteriophage Assembly. (M. Alan R. Liss, Pubi., New York, p. 327

2.

Kozloff, L.M. 1985. In: Proc. of the Second Workshop in Folyl and Antifolyl Polyglutamates. (I. David Goldman, Ed.). Praeger, New York, p. 22.

3.

Maley, G.F., F. Maley. Biophysics 216. 551.

4.

Kozloff, L.M., M. Lute, L.K. Crosby, N. Rao, V.A. Chapman, S.S. DeLong. 1970. J.Virol. jj_«. 726.

5.

Nakamura, K., L.M. Kozloff. 54Q, 313.

6.

Laemmli, U.K.

7.

Towbin, M., T. Stachlin, J. Gordon. Sci. U.S.A. 76. 4350.

8.

Douglas, C.G., B.F. King. 75. 333.

9.

Purohit, S., R.K. Bestwick, G.W. Lasser, C.M. Gogers, C.K. Mathews. 1981. Gene.J.Biol.Chem. 256. 9121.

1970.

1982.

Arch, Biochem. &

1978.

Nature

Biochem. Biophys.

Acta

227, 680.

1984.

197y.

Proc. Nat. Acad.

J. Immunol.

Methods

10.

Howell, E.E., J.E. Villafranca, M.S. Warren, S.V. Oatley, J. Kraut. 1986. Science. 231. 1123.

11.

Kozloff, L.M., M. Lute.

12.

Sadewasser, D.A. L.M. Kozloff. Biophy.Res.Comm. 116. 1119.

1981.

J.Virol. 1983.

40. 645.

Biochem.

EFFECTS OF POLYGLUTAMYIATTON ON FOLATE COFACTOR AND ANTTFOIATE ACTIVITY IN THE THYMIDYLATE SYNTHASE CYCLE OF PERMEABILIZED MURINE LEUKEMIA L1210 CELI£

Thomas I. Kaiman Departments of Medicinal Chemistry and Biochemical Pharmacology, State University of New York, Buffalo, NY 14260

Introduction Polyglutamylation of folate cofactors and cytotoxic folate analogues is an important metabolic process (1,2).

The essential function of the enzyme,

folylpolyglutamate synthetase, responsible for building the poly-y-glutamyl chain was demonstrated by the finding that mutant mammalian cells lacking this enzyme activity cannot survive, unless endproducts of folate requiring metabolic pathways are supplied (2,3). Explanations advanced for the vital role of polyglutamylation (1,2) include increased cellular retention of polyglutamylated folates. Another, based on work with isolated enzymes, is the strong preference of many folate dependent enzymes for the polyglutamylated forms of their folate cofactors over the corresponding monoglutamates (lower K^ and/or increased V^^).

In our laboratory, we have used permeabilized L1210 leukemia

cells to study the effects of polyglutamylation on cofactor and antifolate activity in thymidylate biosynthesis.

Results Partial permeabilization of L1210 cells using high nolecular weight dextran sulfate (5) permitted the study of the influence of polyglutamates on the thymidylate synthase cycle (TS cycle) composed of thymidylate synthase (TS), dihydrofolate reductase (DHFR) and serine hydroxymethyl transferase (SHMT), outlined in Fig. 1. With the provision of substrates and cofactors the TS cycle is fully operational.

The cycle can be initiated at any of the 3

enzymatic reactions and quantitated by measuring the tritium released from the labelled substrate in a manner described for the assay of TS activity in intact L1210 cells (6).

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

764

5-3H-dUMP

1 Thymidylate synthase 2 Dihydrofolate reductase 3 Serine hydroxymethyl

transferase

CONCENTRATION, H Fig. 1. The Thymidylate Synthase Cycle and the Permeabilized Cellular Enzyme Assay. Fig. 2. Cofactor Activities of Pteroylhexaglutamate and Pteroylglutamate (Folate) in the TS Cycle of Permeabilized L1210 Cells. A comparison of the cofactor activities of the mono- and the hexaglutamate forms of folate shown in Fig. 2 clearly indicate a marked preference of the TS cycle for the hexaglutamate. At the physiologically relevant concentration range of 1-3 iiM, maximal activity is achieved by the hexaglutamate, whereas the monoglutamate gives only negligible activity. When SHMT was excluded from the cycle by replacement of serine with formaldehyde as one-carbon source for the 5,10-CH2H4PteGlun cofactor, a similar pattern was obtained, but the difference was less pronounced, demonstrating that both SHMT and TS contribute to the observed effect. Kinetic analysis of the data showed that both the apparent K m and the apparent V ^ ^ are affected by the extra glutamate residues. The results strongly suggest that even full intracellular retention of the monoglutamates of folates would not be sufficient to support effective DNAthymine biosynthesis in L1210 cells in vivo.

765 Hie effects of the extent of polyglutamylation on the functioning of the TS cycle were also examined by varying the chain length from 1 to 7. Fig. 3 illustrates the effect of chain length on the cofactor efficiencies of pteroylglutamates expressed as the apparent V^JJ/K • The results demonstrate the superior effectiveness of the polyglutamate forms.

The differences are

most pronounced between the monoglutamate and the di- and triglutamates with the efficiencies reaching a plateau at 4 glutamate residues. eo MTX[Glun]

50

pM

V/K,

40

20

-

Y/A> j) a

7rrX////)t?}?\

Fig. 3. Chain Length Dependence of the Cofactor Efficiencies of Pteroylglutamates in the OS Cycle of L1210 Cells. Fig. 4. Chain Length Dependence of the Thymidylate Synthase Inhibitory Activity of Methotrexate Polyglutamates in Permeabilized L1210 Cells. The direct inhibition of TS by methotrexate (Mtx) and its polyglutamates was also investigated in this system. As shown in Fig. 4, the inhibitory activity of Mtx increased 60-fold by 3 additional glutamate residues, but no further increase was evident up to the heptaglutamate.

In contrast, the quinazoline

antifolate CB 3717 (7), a potent TS inhibitor, reached stoichiometric inhibition at the triglutamate level at a concentration 15-fold lower than the IC^0-value of the monoglutamate.

This represents a 3-orders of magnitude

higher potency than that shown by the polyglutamates of Mtx.

766

Conclusions This work demonstrates that permeabilized cellular assay systems are useful for the study of the effects of folate and antifolate polyglutamates on intact metabolic pathways. The study of the TS cycle of permeabilized L1210 cells revealed that polyglutamylation of folate cofactors is essential for thymidylate biosynthesis.

The results provide a functional connection between

the genetic evidence for the vital role of folylpolyglutamate synthetase and the preference of many isolated enzymes for the polyglutamylated forms of their folate cofactors.

Acknowledgement This work was supported by research grants CH-192 from the American Cancer Society and CA 35212 from the National Cancer Institute.

References 1. Goldman, I.D., B.A. Chabner and J.R. Bertino, eds. 1983. Folyl and Antifolyl Polyglutamates. Plenum, New York. 2. Goldman, I.D., ed. 1985. Proceedings of the Second Workshop on Folyl and Antifolyl Polyglutamates. Praeger, New York. 3. McBurney, M.W. and G.F. Whitmore. 1974. Cell 2, 173; 183. 4. Taylor, R.T. and M.L. Hanna. 1975. Arch. Biochem. Biophys. 171, 507. 5. Kucera, R. and H. Paulus. 1982. Arch. Biochem. Biophys. 214, 102. 6. Yalowich, J.C. and T.I. Kalman. 1985. Biochem. Pharmacol. 34, 2319. 7. Jones, T.R., A.H. Calvert, A.L. Jackman, S.J. Brown, M. Jones and K.R. Harrap. 1981. Eur. J. Cancer 17, 11.

SUMMARY R. E. MacKenzie McGill University, Department of Biochemistry, Montreal, Quebec, Canada H3G 1Y6 Pteroylpolyglutamates

are preferred substrates for many enzymes having much

lower

than

values

effects clear

of

Km

seen on V^,

the

corresponding

While this f a c t

that we have demonstrated a l l

provide,

and e s p e c i a l l y

if

they

i s well

monoglutamates, established,

the advantages

serve

it

that

a regulatory

with is

still

these

role.

lesser not

substrates

Instances

of

channeling of intermediates between c a t a l y t i c centers have been documented, and a major question

is

whether

this

part of the problem includes

integral

phenomenon occurs the potential

more widely.

enzyme-enzyme associa-

t i o n that has been proposed for many metabolic pathways i n vivo. concept

is

potential role,

not

unique

feature

perhaps

to

folate-mediated

metabolism,

provided by the polyglutamates in

maintaining

specific

is

An

While the

the

that of

additional

a

protein-protein

structural

interaction.

Precedent exists from the observation that the polyglutamates bind to other proteins,

such as hemoglobin, strengthen the quaternary structures of some

enzymes, and in the best-docmented and most elegant studies, have been shown to be important structural components associated with the proteins of the T4 baseplate assembly. To demonstrate

the

types

of

roles

postulated

for

with associating enzyme systems i s extremely d i f f i c u l t .

the

polyglutamates

This i s true both

to i s o l a t e or reconstitute such putative complexes as well as to assay in a meaningful high

fashion

concentrations

permeabilized

maintained

mammalian

exogenous substrates amates.

so as to distinguish

cells

to

in assay

specific

"channeling"

microenvironments. certain

pathways

from simple The

"in

use

situ"

has c l e a r l y demonstrated the requirement for

of with

polyglut-

This approach holds some promise for future studies in this area.

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

RECENT ADVANCES IN THE STUDY OF DIHYDROFOLATE

REDUCTASE

Raymond L. Blakley and James R. Appleman Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee

38101

Introduction

Recently

information

about

dihydrofolate

(DHFR) has been accumulating

rapidly.

Striking

obtained

Refined

results crystal

have

been

structures

have

DHFR from several sources: of

Escherichia

coli

DHFR

Lactobacillus casei triazine

binary

complex

(1);

with with

ternary

chicken

ternary

DHFR

DHFR

of

consists

of

secondary cutting

structure.

hydrophobic DHFR.

one

side

and

quite

trimethoprim

the

DHFR

with

chains

E.

of witn

the

The

coli

DHFR

site

the the

that

of

of

groove

of

join

the

the

exception

It of

is

elements lined

Asp-27

B-sheet.

The

(E^ in

in an extended

stretches

nicotinamide

Chemistry and Biology of Reridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

of

deep with coli

chicken

conforma-

across

moiety

are

structure

is a cavity about 15A

structure.

that

(Figure

containing

Four a helices

remainder

to all three enzymes

a shallow

strands

ternary

(4).

structure

in the case of bacterial enzymes or Glu-30

NADPH Dinds

a

binary

trimetho-

is a twisted B sheet

large,

The active face

tion, occupying five

the

(3); and

the same basic backbone

some

of

(5,6).

8-sheet

loops,

across

numbering)

the

of

complex

complex

(2);

chicken

eight strands, all parallel except the last. against

complexes

binary

complex of NADPH and

liver

1) in which the principle feature packed

for

(MTX)

NADPH-MTX

liver

1.5.1.3)

crystallographers.

reported

inhibitors with

complexes

three enzymes have

(EC

coli DHFR (3); the ternary

have also been described All

the

chicken

of eight other

and

the

the methotrexate

trimethoprim complex with complexes

by

DHFR (1); the ternary

innibitor

prim-NADPH

been

reductase

is

the

ends

located

770

Figure

1 .

Lactobacillus

and m e t h o t r e x a t e . to

tiis-4i> a n d

f r o m Kef. in

the

entrance

side

of

and

chains.

the

coli.

hydrophobic

moieties

hydrogen

carboxamide boxyls,

complex

with

indicate A r g - 4 4 a n d Thr-116,

in

or van der waals

and n i c o t i n a m i d e many

ternary

UHFK

Reproduced

NADPH

equivalent

with

permission

1«.

hydrophobic and

Arrows

Thr-113

casei

uonds

to polar to

bound

of

MADFh with

from

the

side chains, water

cavity.

interactions

There

are

numerous

of the adenine, amino

phosphates,

acid

side

adenine

ribose chains, and

to back bone amide Ns and

molecules

that

form

bridges

to

the carpolar

771 Ail

inhibitors

tnidine,

bind w i t h the 2 , 4 - d i a m i n o

^-triazine

or

pteridine)

h e t e r o c y c l i c ring

similarly

positioned

(pyriin

the

a c t i v e site cavity so that N-1 and the 2 - a m i n o group are close Asp-27 the tion the

(Glu-30

existence

of

(Figure 4-amino

in c h i c k e n

DHFR)

two h y d r o g e n

2).

with a

and

two

consistent

bonds and a c h a r g e - c h a r g e

In addition,

group

geometry

there

backbone

are

hydrogen

carboxyl

the 2 - a m i n o group and a bound w a t e r m o l e c u l e w h i c h bonds to o x y g e n or a c o n s e r v e d Thr residue. otrexate

between

and

between

also

hydrogen

In the case of m e t h -

(MTX) N - 8 is also h y d r o g e n bonded to a b o u n d w a t e r

in turn is h y d r o g e n and

van

heterocyclic

der

Waals

In all cases n u m e r o u s

interactions

ring and side

chain of

also

the

cooperativity

tors

is

mide

inoiety

in part due

side

chain

of of

to some

bound the

in the NADPH

bound

binding

direct and

occur

of

inhibitor

the chain

residues.

NADPH

interaction

its

hydro-

between

inhibitor and m a i n

and side c h a i n a t o m s of n u m e r o u s amino a c i d Positive

which

bonded to the active site carboxyl and to the

h e t e r o c y c l i c N of a c o n s e r v e d T r p . phobic

interac-

bonds

oxygens

to

with

of

associated (3,6).

and most the

ribose

Another

inhibi-

nicotinawith

the

important

c o n s e q u e n c e of the p r o x i m i t y of bound N A D P H and inhibitor

is

that

R

H

ILE 5

H

l

. 0 —

405

THRII3

TYR III

WT- MTX Figure E. coli

2.

Probable

hydrogen

bondings

DHFR. R e p r o d u c e d w i t h p e r m i s s i o n

1y«6 by A A A S .

between

methotrexate

from Ref.

20;

and

copyright

772 binding that

of

are

NADPH

probably

otherwise

expels

bound

in

seven

the

lower

to

nine

part

of

enhance

its b i n d i n g .

a m a r k e d change joining

cavity

of a t t a c h e d

(6).

and

side

This

loop

its change chains

of p a r t of forms

one

in c o n f o r m a t i o n

bringing

some

The

trimethoprim

been

inhibitor

(3).

It

particularly DHFR,

very

favorable

binding that

to bound

inhibitors)

to aB where

backbone the

significant Matthews

with

the

less

its

and

is

4-amino instead

E.

coli

al.

have

ring

usual of

of to

E.

bound

tri-

trimethoprim

binding

(and

other

geometry

approximately

hydrogen two.

bond

to

of

they

so 1 A

only

Although

binding

holoenzymes,

recently

is

it m a k e s ( a

trimethoprim

can

for t r i m e t h o p r i m s e l e c t i v i t y

et

chicken

factors

where

a position

the

DHFR

formed

from

above

aspect

an a l t e r e d

of

inter-

the t e r n a r y complex of

that

in the g e o m e t r y

position

bacterial

and

the

benzene

group

the

in

loop

hydro-

complexes

(3,6)

favorable

(4) have

to

ternary of

the

favorable

a position

ring o c c u p i e s

carbonyl

chicKen

third

into

holoenzyme

are other d i f f e r e n c e s to

moved

Another

the n e t e r o c y c l i c

closer

the

in the case of

interaction

chicken

the

from E. coli

that

being

(3,6).

of

comparing

DHFR

appears

Met-20

methoprim

by

with

favorable

coli

weakly

binding

investigated

this

liver

tight

of

alters

into m o r e

chain.

selectively

the flexible

side

a c t i o n s w i t h the i n h i b i t o r ' s side

one

site

Finally, binding of the c o f a c t o r results

in the c o n f o r m a t i o n

8A and a B

phobic

by

molecules

active

This c o f a c t o r - i n d u c e d d e s o l v a t i o n of the inhibitor may well

(3).

has

water the

there

trimethoprim

seem

not

to

be

(3,4).

reported

(7)

crystallographic

r e s u l t s on DHFR of a quite d i f f e r e n t s t r u c t u r e , a type II R p l a s mid-specified bition =

90

somal

by uM)

despite

E ^ coli

78 amino tion.

This enzyme

The

a

DHFR.

acid

and

Km

of

adjacent

the

H 2 folate

each

surfaces

together barrel

is c o m p l e t e l y very

weakly close

of

folded the

in

three

a

r e s i s t a n t to

inhibited to

It is a d i m e r i c m o l e c u l e

that with

beta

by

interface.

between

corresponding

strand

beta

for

of

subunit

two

identical conforma-

longest

beta

strands

runs

D in s u b u n i t II,

(K^

chromo-

barrel

An 8 A g r o o v e strand

inhi-

MTX

form a third beta barrel having six

the p r o t o m e r - p r o t o m e r

length

is

for

subunits,

outer

each m o n o m e r at

DHFR.

trimethoprim

and

it

is

in

strands the

full

I and

the

proposed

773 that

NADPH

binds

in

this groove.

ternary

complex with NADPH and

suggest

that

with

type

similar

II R-plasmids

catalytic

A hypothetical

H 2 folate have

machinery

model

is proposed

independently

to

that

of

for

which evolved

the

the

would DHFR

chromosomal

enzyme.

Nuclear Magnetic Resonance

Complexes alogues

of

L.

exist

casei

The

NMR

nicotinamide surface

(8,9)

evidence

into solution

enzyme

methoprim

trimethoprim of

NADP+

and

two conformations

that slowly

suggests

or

in

one

in

with

concomitant

the other

changes

undergoes

conformation.

"ring

flipping"

The

the

in the ring

benzyl

s-1 )

(65

at

conformation

By contrast the nicotinamide

(9).

an-

(6 sec - 1

interconvert

that

its

(8) in approxi-

ring of the coenzyme has swung away from

phate moiety the

with

as a mixture

mately equal amounts 31°).

Studies

the

enzyme

pyrophos-

is bound ring of

(10).

to

tri-

Similarly

there are three interconvertiny conformational states of the complex of L. casei DHFR with folate and NADP+

(11).

In

casei

3',5'-difluoromethotrexate

benzoyl

ring

of

the

bound

axis at a rate of 7.3 x 10 3 approximately to

rotation

estimated

bound

inhibitor s-1

to

L.

"flips"

at 25°C with

DHFR

about AHf

(12)

its

symmetry

11.5 kcal/mol,

two-thirds of which is due to the intrinsic about

the

contribution

bonds of

at

the

C-l'

and

protein

C-4'.

to

the

the

The

barrier

rather

observed

low

barrier

(=4.8 Cal/mol) implies that the binding site for the benzoyl ring is not rigid

and

flipping

is facilitated

by

"relaxation"

of

the

protein, ie small transient displacements of protein atoms.

Resonances

have

casei DHPR (13).

been

assigned

to

32 of

the

These residues are widely

out the structure of the protein.

162 residues distributed

of

through-

Assignments have been made for

774 complexes

with

MTX,

witn MTX

C o m p a r i s o n of a s s i g n e d plexes

indicates

coenzyme 13-23)

but

there

structure

in a - h e l i x form

many

in s p e c t r a of

are

in the loop c o n n e c t i n g and

firms

resonances

and w i t h

the

C

sides

earlier

of

studies

three

coenzyme-induced

the

44-49).

These

hydrophobic

(e.g.

14,15)

by

conformation (residues

regions

cavity.

which

com-

is u n a f f e c t e d

8 - s t r a n d A and a - h e l i x B

(residues

NADP+.

MTX and

these

that the B - s h e e t of tne p r o t e i n

binding

changes

and N A D P H ,

the

of

This

con-

the

cer-

lacked

tainty that the s p e c i f i c a s s i g n m e n t s p e r m i t

in the p r e s e n t

work.

That

bacterial

verte-

N—1

of

inhibitors

bound

to

DHFR

from

and

brate sources remains p r o t o n a t e d o v e r the a c c e s s i b l e pH range been confirmed

(16,17).

Oligonucleotide-Directed

Mutagenesis

This

is a new m e t h o d of studying

ship

in e n z y m e s .

tions

into

Figure

E^

1).

DHFR

was

Chen et al.

coli

Both

conformation. intrinsic

enzyme

DHFR

3).

Km

for

for

Gln-45

DHFR:

make

by

(18)

introduced +

have

of NADPH

fluorescence. KQ for

NADPH

These

Gin

and

restricted, measurement

Changes higher

adenine results

in

than

relation-

two s p e c i f i c Thr-113

*

local

to the w i l d - t y p e

stopped-flow

acetylpyridine DHFR.

the s t r u c t u r e - f u n c t i o n

His-45

replacements

Binding

studied

Gln-45

of

kon

crystallographic

m o d e l , does

than a H - b o n d .

This

water

crystal

in

is stronger

the

than w i t h

effects

quenching and

on

Gln-45

k oj =f

of for

wt

DHFR

(Figure

dinucleotide

is

also

greater

not p r o m o t e

is c o n s i s t e n t w i t h Gln-45.

(see

for

indicate

structure.

muta-

Val

(wt) and

that

the

salt

b e t w e e n His-4 5 and the 2 ' - p h o s p h a t e of N A D P H , p o s t u l a t e d

as

has

b e t t e r binding of H i s - 4 5 being

However,

either

bridge

from

the

NADPH

exposed

to

interaction

775

kon

His-45 DHFR

k„.

G l n - 4 5 DHFR

I

*

-O-

_I

2

k 0 ff G l n - 4 5 DHFR

D

I

—k ff fH i s - 4—5

I

0

DHFR

7

0

8

10

PH

Figure

3.

pH

dependence

wc and Gln-45

of

Kon

and

kQff

for

NADPH

binding

to

coli DHFR. Reproduced with permission from Ref.

1 a. Replacement of Thr-113 network, involving 25-rold of

the

and

dependence DHFR.

K.u tor

ertect of

This

is

(thought to have its hydroxyl in an H-bond

H 2 folate) the

due

V and

suggests

pK a -

can

also

loss account

of for

Vai

i-l'l'A binary

V/Kln

are

that

removal

increases

complex

to a faster

and Asp-27 destabilizes its

with

0.4

K.m for

20

to

30-fold.

off

rate.

The

pH

units

lower

of

H-bonding

pK.as than

between

the acid form of Asp-27 with this

the

single

effect

hydrogen

on K m

for

H 2 folate

bond

pH

for

wt

Thr-113

lowering

(2-3

ti2 folate

All

from

of

kcal/mol) and

Kq

for

HTX. Since

neither

113) must

not

change alters V the mutated be

involved

binding of the substrates.

in the

residues

catalytic

process

(His-45, Thrbut

only

in

776 Two

other

franca acid

mutants

et al.

of

(19) in which

(Asp-27)

is

is a revertant

essential

hydride

(Figure reduced which

tor the

4), by

has

the

4.

27 m u t a n t

DHFK

that rate

profiles for (A).

in

of

For

fact

In

as

the

the bound

(a)

log

case

pH

further

from Kef. 2U by p e r m i s s i o n ;

the

The read-

pH

and

is

wt

wt

(b)

(20) is

enzyme, appar-

even at a pH

log

k^t-/!^

(o) and

see R e f e r e n c e

copyright

not

lowered

H2folate

the

DHFK

is

DHFR

7, A s p - 2 7

substrate,

kcat

details

of

oligo-

Asn.

of

6 and

(x), A s n - 2 7 m u t a n t

by

of A s p - 2 7

that

Villa-

low, but

preprotonated

between

of

with

have

carboxylate

only

V/K

wt DhFK

Asp-27

by

carboxylic

constructed

approaches

enzymes.

V and

obtained

active site

replaced the

that

a protonation

pti

(dihydrotolate)

duced

mutant

a maximum

been

both mutants

transfer.

suggesting

ently m e d i a t e s

Figure

so

catalytic

had

mutation,

to Ser-27.

activity

5

One

mutagenesis,

ily m e a s u r a b l e towards

L)HFR

the invariant,

replaced.

nucleotide-directed other

t,. coli

20.

1y86 by A A A S .

Ser-

Repro-

777 w h e n little p r o t o n a t e d H 2 f o l a t e Asp-27

is n o t e s s e n t i a l

bound H2folate. low

pH.

is

consistent

folate

tonated folate

is less

with

in solution.

subsequent

M u t a n t DHFRs are unable

This

for N - 5 of

is p r e s e n t

for e v e n t s

this

interpretation

Km

for

H 2 folate

the

MTX Dinary

and

comparisons

(20).

The

chain

or

bound

MTX.

with

the

no

the

is

for

nearly

positions

of

side

this

the

of

featureless close

unmutated

the

in wt e n z y m e .

The

of

(0.9 A). of

to

H-bonding

to A s p - 2 7

wat-403,

in

arrangement

of

to

(Figure

turn the

to

tne

by

UV d i f f e r e n c e

showed

that

5).

moves

shown

even

amide

mutant

1.4

enzymes

is

spectra

MTX

DHFRs

bound

to

of

is in

compared

H-bonded

again

because

of

the

re-

network.

As

expected,

MTX

and

(21).

wt

at 13

by

C

neutral NMR

The

enzyme

pH

as

spectra

of

latter

technique

remains

even

down

(5.7).

The m o s t likely H - b o n d i n g systems for interaction of

tonated

MTX

Asn-27 the

enzyme

Asn-27

elimination

are

enzyme of

the

wt

enzyme,

and

times

only

27

interaction

The

Ser-27

for

reveals no c o o r d i n a t e

pKa

higher

of

than

sacrifices

minus

wt

unpro-

free

MTX

K Q for

Since

DHFR-MTX

changes g r e a t e r

the

remains

unprotonated

is

energy.

map

of

similar.

kcal per mol of binding difference

below

remarkably ionic

enzymes

protonated

tonated

with

the m u t a n t well

to

is

pH, M T X D o u n d

4.5,

the with

hign

pH

the

identical

at

to

to

or

This is due

Asn-27

unprotonated (20)

the

which

A

bonding

to the m u t a n t

whereas

group

Fixed W a t - 5 6 7 ,

by

hydrogen

bound

[ 2 - 1 3 C]MTX D o u n d

the

A

backbone

change

different

1.9

binding

the

chains

from

in

MTX

a position

largest

for

DHFR-MTX

except

to

side

occupies

at

Asn-27

in

KQ

clear

refined

map

conformation

chain

in

became

p o s i t i o n of fixed w a t e r m o l e c u l e W a t - 4 0 3 bonding

pro-

increase

increase

structures

molecules in

that of A s p - 2 7

marked

difference

change

The A s n - 2 7

more

x-ray

water

fixed

is

in

pKa

no

DHFR.

reasons

density

DHFR-MTX

two

There

even The

of

electron

wild-type

site.

an

complex.

elegant

of

since

is p r e s e n t in s o l u t i o n even at pH 5, so no p r e p r o -

S u b s t i t u t i o n of Asn or Ser for Asp-27 c a u s e s a m a r k e d

vicinity

of

to reduce folate even at

than - 1 . 5 , so that e s s e n t i a l l y

t o n a t e d folate can bind to m u t a n t

minus

However,

to p r o t o n a t i o n

pro-

with

the

binding

for

wt

only

the

to

enzyme,

about

DHFR-MTX

than 0.2 A in

MTX

1.8

again

backbone

778 chain, of

uninutated

(UD1 )

Asp-27

of

Wat-UU5, by

side

i>er-2/ o c c u p i e s

a

takes

serine

binding

in

the p l a c e

going

or H'i'A. just

wild-type

hydrate.

in

enzyme:

chains

a position

from

DHt'K,

of 0D2.

There wt

However,

below

where

and

a

the g a m m a

oxygen

the

oxygen

new

delta

water

In effect, A s p - 2 7

are

three

binary

important

complex

to

molecule,

is

replaced

changes

that

of

in

the

loss of ionic i n t e r a c t i o n b e t w e e n N-1 and the side

chain

of r e s i d u e 27; loss of a h y d r o g e n bond b e t w e e n r e s i d u e 27 and Z-ainino

group;

2-amino

group

interaction energy kcal

(as

in the must gap

it is e v i d e n t combination for

creation the

accounts

inol"1

der Waal's

tool

and and

for

case

enzyme.

1.6 k c a l the

bond

will

crystallography, the

role

undoubtedly

of be

the in

and

the 3000-fold

that the use of d i r e c t e d m u t a g e n e s i s , x-ray

between

exploited

the

binding the

2.6 van

increase

in

especially

in

is an extremely

structural

the

ionic

an a d d i t i o n a l

hydrogen

for

gap

loss of

decrease

enzyme),

lost

to a c c o u n t

If

per m o l

the A s n - 2 7

from

in order

with it

a

of

result

determining

enzyme.

of a van der Waal's

surrounding

MTX

Ser-27

powerful

features much

more

of

the

in

the

future. R

R

HE 5

H

H

H

ASN 27 MTX

Figure

5.

mutant

OtiFK

HE 5

H

Probable (left)

SER 27 MTX hydrogen and

Ser-27

bondings mutant

trom Kef. 20 by p e r m i s s i o n ; c o p y r i g h t

between DtiFK

MTX

(right).

1986 by A A A S .

and

Asn-27

Reproduced

779 Stopped-Flow Spectrophotometry and Fluorimetry

Cayley et al. (22) obtained evidence that wild-type E. coli DHFR exists as an equilibrium between ers),

only

folate.

one

of

which

is

two forms

able

to

(presumably

bind

NADPH,

conform-

H 2 folate

or

MTX and trimethoprim are able to bind to both forms.

In

stopped-flow measurements of the rate of quenching of the protein intrinsic

fluorescence

by mixing

with

ligand,

analyzed as the sum of a rapid process the binding

the decay

(reaction of ligand with

conformer) and a slower process

(conversion

non-binding conformer to the binding conformer). al.

(23) nad obtained

curve

similar evidence

of the

Earlier Dunn et

that L^ casei DHFR also

exists as an equilibrium mixture of two forms, only one of which can bind NADPH. Penner and catalyzed

Frieden by

substrates, the reaction substrate.

(24) recently

coli

DHFR

tne reaction

rate

is abolished

showed

is started

that when

by

adding

increases with

by preincubation

Preincubation

about twice that obtained

also

results

the

reaction

enzyme

time.

to

of DHFR with

in an

initial

for the non-preincubated

the

The lag in either

velocity

enzyme.

The

half-time for the increase in velocity observed with nonpreincubated enzyme is about 0.9s, which corresponds to the half-time of the conversion of the non-binding form of the enzyme to the binding form. We

have

performed

similar

casei, Streptococcus

with

DHFR

faecium and bovine

studies

liver

from

similar results to Penner and Frieden with E. coli. the

other

two

bacterial

enzymes

were

col i, L.

(25) and obtained Results with

qualitatively

similar

(Figure 6) except that the fraction of the enzyme in the ligandbinding

form was less.

(pH 7.5, 20°C) , 58% of

We estimated E. coli

that under our conditions

DHFR was

in

the

ligand-binding

form, 40% of S. faecium DHFR, and 9% of L. casei DHFR.

No lag in

reaction was found when bovine DHFR was added last to substrates, and initial velocity was no greater when the enzyme was preincubated with either substrate.

780

0

10

20

30

Time

Figure casei

6.

Substrate

DHFR.

preincubated final

substrate.

The

pg/ml),

50

50

yM

NADPH,

Equilibria case

of

Ki

enzyme

inhibitor

value

the

(26),

the

binding

of

and

chicken

liver

enzyme

by

taining over

of

50

mM

Tris

of

both

of

L.

H2folate

or

DHFR

(8.8

buffer,

500

to o c c u r

in

If

to

The

and

in

is

inhibitor,

inhibition" which

that

and

this

mM

DHFR

(Figure

is

enzyme or

in

added

com-

a to

E.

rate

to

coli

of

was the

tight-binding a mixture

velocity

"recovery to

con-

decreases

obtained

last

his

sudies

equilibria the

of

last

From data

experiments, H2 folate

the in

presence the

their

S^ faecium,

for

changes

7).

overall

with

by M o r r i s o n a n d

from

added

the

vertebrate

occurs

extended

evidence

the

enzyme

minutes

bacterial

first presented

time-dependent

reaction

in

also appear

Evidence

inhibitors

MTX.

experiments

activity

NADPH,

contained:

subsequently

(27-29).

substrates

"progress ty"

have

measuring

a period

of

DHFR was

who

many

like

the

h a v e a p r o f o u n d e f f e c t on the

inhibitor.

catalytic

inhibitor

H2folate,

complexes

p l e x e s of Sj_ f a e c i u m colleagues

mixture

conformers

these e q u i l i b r i a

for

obtained

pM

in

with

20°C.

between

D H F R s , and

hysteresis

was

without

KCl at p H 7 . 3 5 ,

(s)

induced

Enzyme

40

in

of the

such

activiother

781 components,

it

corresponds

to

forward the

and

is

possible

the

initial

compute:

binding

rate constants

reverse

complex;

corresponding

to

initial and

Ki*

to inhibition

the

initial

reaction;

kf

K^,

and

which

kr

the

for the slow isomerization of

the

after

final

inhibition

constant

the slow isomerization

is com-

plete .

K

K E +

MTX + H

+

.

""

f

- E.MTX.H"\

-

E.MTX.H+*

off Kn

(initial)

V K

The

equilibrium

tion

reaction

11.9

min-1 ,

min).

constant

ranges and

The

Clearly, resulting

(kf/k r

from

kr

can

ratio

development

of

the

as

that

many

conventional

low

kr/kf+kr

initial

inhibition

with

=

for a routine

Kiso)

for

1.1 to 747, kf has be

the final inhibition from

(final)

d

as

of

min-1

the

0.33

(ti/2

ratio

requires

of

inhibitor,

quite

a long

spectrophotometric

Ki*/Kj..

of

tne

and

inhibition

of

that

the

period

assay.

to 116

is often very much lower than

binding

estimates

isomeriza-

values

0.006

gives

the

full

compared

Consequently DHFR

by

anti-

folates produce much too high a value of KiWe have also studied conformational

isomerizations

and

DHFR

ternary

flow

inhibitor

measurements

of

binding.

In our

was

to occur

found

complexes

fluorescence

initial

collaboration

by

with

the

quenching

studies MTX binding

in two steps, a rapid

subsequent isomerization in

of

(30,31).

Dr.

James

of

the

use of due

binary

stopped-

to

ligand

to S. faecium

initial

binding

DHFR and

Recent results with human Freisheim

(32) have

shown

a

DHFR that

binary complex formation with this enzyme also involves an isomerization step following the

human

enzyme

kon

initial complex formation is

extremely

high

and

the

(Table 1). initial

Ki

For (=

782

0

1

2

3 Time

6

(min)

7.

DHFR.

DHFR added last to a mixture with final composition:

DHFR,

90

uM

NADPH,

50 mM Tris,

76

uM

of

5

Figure

shown,

Slow development

4

inhibition

H 2 folate,

25 mM acetate,

MTX

by MTX of at

the

25 mM MES,

S.

faecium 6 nM

concentrations

100 mM NaCl

at pH

7.4, 20°C.

k0ff/K0n)

is

correspondingly

low.

We

have

also

performed

a

preliminary examination of the formation and isomerization of the ternary not K0ff

complex

altered

of

oy

is decreased

initial mined

Ki.

human

the

DHFR

presence

100-fold

Although

(Table of

with

k.on

and

I).

NADPH

in

The

value

the

active

a corresponding k0ff

for enzyme from other sources,

have

not

For all four enzymes

zation ther than case

of

to the of

the

give

initial a

final

initial human

large enough all binding

Ki. as

K^

complex that

Although

the

been

DHFR

lowers

decreases is is

from

deter-

the

40

to

not

other

K^

400 as

still times

great

in

the

over-

DHFR.

it

furlower

that this change exerts a major effect on the of MTX by human

sources

initial isomeri-

still

release

for

lowering of yet

is

and

DHFR

ternary

is but

for which data are available

value

kon

site

in two other cases the pre-

sence of NADPH in the active site drastically Ki.

of

Thus, after

com-

783 piete equilibration of the ternary complex, release of MTX and its replacement by another ligand such as H2folate is limited by the rate of reversal of the isomerization step with t\/2 of 5.9 min (at 20" and pH 7.5). Although the evidence is still fragmentary it appears likely that the binary and ternary complexes of tight-binding inhibitors like H'i'X with IMFK from most sources undergo conformational changes that make a critical contribution to overall binding. The difrerence in conformation of the initial complex and the final

Table 1 .

Kinetics of Methotrexate binding to DHFR

from bacterial and Vertebrate Sources

Binary Complexes Initial Source

¿.coli i-.. casei S. faeciuiii z Chicken liver rluiuan

k

on

H"1 s"1 1.6 x 10 7c x 10 6e 3.0 x 10 5e 2.0 x 1 U8

k

off

s"1 10e 17.2e 0.52e 1

K

D

a

Final K

Db

nM 620c 3000e 1730e

pM 0.62d 4.0e 75e

5

0.11e* = 0,

eclipsed

for

atoms, to

averaging

-1.000

important

these for

atoms N1,

The charges on the endocyclic

an N2,

N1 and

N3 are uniformly more negative than those on N2 and N4, reflecting the formers' greater basicity and proton-binding Table II: Coaparison of the Pyriaidine Ring X-ray Structure, CNDO/2, and MM2P Results. Bonds

X-ray

CNDO/2

MM2p

C6-N1 N1-C2 C2-N3 N3-C4 C4-C5 C5-C6

1 .349 1.332 1.327 1.336 1 .434 1 .410

1 .358 1 .347 1 .347 1 .360 1.356 1 .405

1 .349 1 .340 1 .337 1 .343 1 .400 1 .370

C2-N2 C4-N4 C5-C7 C6-C61

1 .353 1.365 1.555 1 .527

1 .392 1.453 1 .490 1 .494

1 .343 1 .343 1 .548 1 .515

0.047

0 .019

RMSD Torsion

angle

C6-N1-C2 -N3 N1-C2-N3 -C4 C2-N3-C4 -C5 N3-C4-CS -C6 C4-C5-C6 -N1 C5-C6-N1 -C2 RMSD

Angles

Geoaetry DAEP

froa

The

MM2p

used

X-ray

CNDO/2

MM2p

C6-N1-C2 N1-C2-N3 C2-N3-C4 N3-C4-C5 C4-CS-C6 CS-C6-N1

117..0 125..1 117..2 123 .7 111..8 123..5

116 125 114 125 110 123

119. 5 122. 3 117. 2 122. 8 111. 4 121. 8

N1-C2-N2 N3-C4-N4 C4-C5-C7 C5-c6-C61

117..1 112..0 126..2 123..8

117 .4 108 .2 125 .1 126..4

118. 8 112. 2 123. 5 126. 5

1 .9

1 .8

X-ray

CNDO/2

MM2p

-11.3 7.0 6.1 -13.0 8.3 2.7

- 9.1 8.4 3.2 -12.0 10.8 - 1.7

•12.3 6.7 7.2 •13.6 9.8 2.7

2.0

0.9

.9 .2 .4 .5 .9 .5

affinities. method

here

is

that

(4)

and

developed

by

Allinger modified (9)

by

to

aromatic gated

and TT

ed

and

obtain-

Allinger,

from

associated exo-

para-

were

from

apart

conjusystems.

Force-field meters

Rohrer

accommodate

those with endo-

802 cyclic N atoms which were derived based on an analysis of the Xray

crystallographic

calculated torsion

values

angles

structures

for

are

selected

listed

in

listed bond

in Table

lengths,

Table

II

I.

bond

along

The

Mis-

angles,

with

the

and

corre-

sponding observed crystalline and CND0/2-calculated values. MM2p

calculated

pyrimidine

ring

structures

in

nonplanarity

general

in DAMP,

short exocyclic NH2-C bonds observed They are also superior of root-mean-square

accurately DAEP,

and

reflect

DAPP,

in the crystal

(RMSD)

from

the

the

and

the

structures.

to the CND0/2-calculated values

deviation

The

in terms

observed

struc-

tures . Acknowledgement This research was supported by NIH grant no. CA 34714 awarded by the National Cancer

Institute.

References 1.

For a general review of antifolates, see Blaney, J.M., Hansch, C. Silipo, A. Vittoria. 1984. Chem. Rev. 84.»

2.

Pople, J.A., D.L. Beveridge. 1970. Approximate Orbital Theory. McGraw-Hill, New York.

3.

Jaffe', H.H.. 1977. QCPE 315, Exchange, Indiana University.

4.

Allinger, N.L., et al. 1981. QCPE 395, Program Exchange, Indiana University.

Quantum

5.

Welsh, W.J., V. Cody, J.E. Mark, Cancer Biochem. Biophys. ]_, 27.

Zakrzewski.

6.

Welsh, W.J., J.E. Mark, V. Cody, S. Zakrzewski. 1983. In: Chemistry and Biology of Pteridines (J.A. Blair, ed.). Walter de Gruyter, Berlin, 463.

7.

Cody, V., W.J. Welsh, S. Opitz, S. Zakrzewski. 1984. In: QSAR in Design of Bioactive Compounds, Proceedings of the 1st Telesymposium on Medicinal Chemistry. J.R. Prous, 241.

8.

Cody, V. 1986. J. Mol. Graphics 4, 69.

9.

Rohrer, D., private

Quantum

communications.

S.F.

C.

Molecular

Chemistry

Program Chemistry 1983.

BINDING OF INHIBITORS WITH SPIN-LABELED SIDE CHAINS TO DIHYDROFOLATE REDUCTASE (DHFR) FROM SEVERAL SPECIES

R.L. Blakley, R.F. Kulinski, J.R. Appleman Department of Biochemical and Clinical Pharmacology, Research Hospital, Memphis, Itennessee 38101

St.

Jude

Children's

J.R. Piper Drug Synthesis Section, Southern Research Institute, Birmingham, Alabama 35255

Introduction

The use of spin-labeled ligands in the investigation of ligand-enzyme interactions offers a method of investigating the degree to which side chains of bound ligands are immobilized by complex formation, and the degree to which this mobility i-s altered by various factors.

Here we report on the dissocia-

tion constants of complexes of four spin-labeled antifolates with DHFRs from Lactobacillus casei, Streptococcus faecium and bovine liver, and the degree of immobilization of the spin label in these complexes. follows:

4-(2-4-aminopyrimidine-6-ylmethylamino)TEMPO

The ligands used are as ( 1 ), 4-(2,4-diamino-

pteridin-6-ylmethylamino)TEMPO ( 2 ), 4-(metliotrexate-+-amido)TEMPO ( 3 ), and 4-(methotrexate-Y-amido)TEMPO ( 4 ) (Figure 1).

Methods

DHFR was purified from L. casei, S. faecium and bovine liver according to published procedures (1,2,3,4).

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

804

N—• 0

4-(2,4- Diaminopynmidin-6-ylaminomethyl) TEMPO

/

N

.AN HoN

N—•O

N

4-(2,4-Diaminopteridin-6-ylaminomethyl) TEMPO

Figure 1.

Structures of spin-labeled ligands.

The spin-labeled ligands were synthesized by condensing 4-aminoTEMPO with the 6-bromomethyl derivatives of 2,4-diaminopyrimidine and 2,4-diaminopteridine and with y- and a-methyl esters of methotrexate, respectively. For recording EPR spectra, the spin-labeled ligand (100 wM) in 0.5 M KC1 and 015 M potassium phosphate buffer, pH 7.3, was treated with an excess of EHFR

805

(646 pM S. faecium, 663 pM L. casei, 203 yM bovine). mation NADPH was present in excess over DHFR.

Por ternary oomplex for-

EPR spectra were recorded at

23° on a Varian E-104 spectrometer at 9.52 (Hz, 15.4 dB microwave power, 0.8 G and 100 KHz modulation.

Samples contained in 100 &1 capillary pipettes were

placed in a standard quartz EPR tube.

For correction of spectra, spectra were

collected and stored by an E-935 Varian Data Acquisition System (HewlettPackard 1350A graphics translator, 9835B computer).

Titration of spin-labeled

ligand with DHFR was performed by preparing a series of reaction mixtures (volume 100 pi) of the above composition but containing different amounts of DHFR.

K d was calculated by fitting the amplitudes of the h_! peak and the

enzyme concentrations to appropriate equations by the use of an Apple Ilemicrocomputer.

Figure 2. EPR spectra of spin-labeled ligands in presence of excess EHFR.

806 Results Dissociation constants obtained for binary and ternary complexes of the two methotrexate derivatives 3 and 4 were >1 nM for all three enzymes. The ternary complexes of the other two ligands ( 1 and 2 ) ranged from 2.8-31.8 yM. Binary complexes were not examined for these two ligands because it was anticipated that Kq value would be so high tnat measurements of changes in peak amplitude would be difficult. The spin label side chains of

3

and

4

were immobilized only slightly

(Figure 2).

Correlation times were not significantly different in the ternary

and binary

complexes.

This

is in agreement with X-ray crystallography

structures which indicate that the spin label would be outside the hydrophobic cavity.

By contrast, the spin label of

1

and

2

were highly immobilized

(Figure 2), again in agreement with crystallographic structures that indicate the spin label should be inside the active site cavity and would make hydrophobic and van der Waals interactions with hydrophobic residues lining the cavity. This research was supported in part by research grant CA31922 and Cancer Center Support (CORE) grant P30 CA21765 from the National Cancer Institute, Biomedical Research Support Grant SOI RR05584 from the Division of Research Resources and by American Lebanese Syrian Associated Charities.

References

1.

Nixon, P.F., R.L. Blakley.

1968. J. Biol. Chem. 243, 4722.

2.

Blakley, R.L., L. Cocco, R.E. London, T.E. Walker, N.A. Matwiyoff. Biochemistry 17, 2284.

3.

Gocco, L., C. Temple, Jr., J.A. Montgomery, R.E. London, R.L. Blakley. 1981. Biochim. Biophys. Res. Commun. 10£, 413.

4.

Kaufman, B.T.

1974. Methods Enzymol. 34B, 272.

1978.

AFFINITY

LABELING

OF

DIHYDROFOLATE

REDUCTASE

WITH

AN

IODOACETYL

LYSINE

ANALOGUE OF METHOTREXATE

T.J. Delcamp*, A. Rosowsky + , P.L. Smith*, J.E. Wright +

and J.H. Freisheim*

*Department of Biochemistry, Medical College of Ohio, C.S. 10,008, Toledo, Ohio 43699, + Dana-Farber Cancer Institute, Boston, Massachusetts 02115

Introduction Studies

in

recent years

on

dihydrofolate

structural

analogues' possessing

reactive

(MTX)

2),

and

(1,

trimethoprim

(3)

a

reductase

(DHFR)

have

functional

groups

of

acid

sequences

and

closer examination of the

X-ray

crystallographic

(4)

react

The availability of

structures

has

allowed

identity and orientation of the reactive

with respect to inhibitor binding.

that

methotrexate

2,4-diaminodihydrotriazine

covalently with the enzyme purified from various sources. amino

shown

a

residues

This communication describes the covalent

labeling of three DHFRs by one such compound, an iodoacetyl lysine analogue of MTX.

Results and Discussion The

N a -(4-amino-4-deoxy-Nl0-methyl-pteroyl)-N E -(iodoacetyl)-L-lysine

compound

(APA-Lys-IA) (Fig. 1) was synthesized and shown to inactivate I. casei DHFR in apparent covalent to

identify

the

fashion

(2).

In order

labeled residue

[ H]APA-Lys-IA (labeled at HjN-^V

the methylene hydrogens of the was

incubated with

in phosphate experiments

njv

3

molar excess of moiety)

a 4-fold

buffer were

at

pH

U

iodoacetyl casei

7.0.

performed

APA-LYS-IA

DHFR

Similar

with

DHFRs

L 1

purified from chicken liver and an MTX-resistant line.

an initial rapid binding of the compound. to

human

lymphoblastoid

cell

In each case APA-Lys-IA completely inhibited enzyme activity indicating

allow

the

Quantitation

covalent was

incorporation

achieved

by

of

counting

protein after dialysis against 8 M urea.

Incubation for 2 hours was required 1 mol the

inhibitor

radioactivity

Acid hydrolysis

Chemistry and Biology of Fteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

per

mol

enzyme.

associated

with

followed by

amino

808 acid analysis of the covalently-labeled L. casei DHFR revealed a loss of 1 out of

7

histidines

and

both

the

appearance

(Table I).

substituted forms) the chicken

liver

of

carboxymethylhistidine

Carboxymethylcysteine

and human

enzymes.

An

(1-

and

3-

appeared in the case of

increase

in approximately

1

lysine residue that was derived from the compound itself occurred in each of the

compositions.

Finally,

a

difference

UV-VIS

absorbance

spectrum

of

modified human DHFR vs native human DHFR also revealed that 1 mol of APA-LysIA was covalently associated with 1 mol enzyme (Fig. 2).

«111«

ACID CMPOSITIOHS

OF

Tryptic digestion of the modified bac-

«»THE

AND APA-LYS-IA-M0D1FIED DHFRS

terial HUNAN

I . CASFI AMINO

ACID

NATIVE

MODIFIED

NATIVE

HODIFIED

(HHOE)

(NMOI )

(NHOL)

IHWOI )

ASP

17.9

18.9

19.!

THR

12. 4

13.3

6.3

6.6

SER

3.9

3.9

12.0

12.6

GLU

15.2

17.2

21.4

20.1

7.6

8.9

11.5

9.1

GLY

10.1

11.7

13.»

14.1

ALA

13.7

15.0

5.6

5.7

CYS

-

--

-

--

VAL

12.6

12.8

12.1

12.0

BET

1.9

2.5

6.0

5.9

ILE

5.0

OF

NATIVE

ACID.

AND 1)

MODIFIED

NATIVE.

3) MODIFIED DHFR.

2)

crystal casei

sources.

differences sites The

structure

of X-ray

of

MTX-NADPH-DHFR

J.. (5)

809 shows that histidine-28

interacts closely with the y-carboxyl

Reaction

with

of

APA-Lys-IA

L.

casei

DHFR

is

thus

not

group of MTX.

unexpected.

In

contrast, X-ray crystallographic studies of chicken liver DHFR show that the single

cysteine

residue

at position

11

in

the

N-terminal

participate directly in inhibitor or NADPH binding (6). free APA-Lys-IA was

in solution

investigated

by

is responsible for the

incubating

large

molar

region

does

labeling of the cysteine

excesses

(50

to

iodoacetic acid for one hour with native human and chicken

100-fold)

liver DHFR.

effect on enzyme activity was observed nor was carboxymethlycysteine upon

amino

sufficient

acid length

analysis. and

Thus,

APA-Lys-IA

conformational

may

flexibility

not

The possibility that

possess to

a

extend

No

detected

side-chain away

of

from

of the

active site in order to react with the cysteine residue of either the human or avian

enzyme.

functional

Further

studies

with

MTX

analogues

possessing

reactive

groups may contribute a better understanding of the disposition in

solution of the reactive groups of DHFRs both near and distant from the active site.

Acknowledgment This research was supported in part by NIH grant number CA-41461 (J.H.F.) and in part by CA-25394 (A.R.)

References 1.

Johanson, R.A. and J. Henkin.

1985.

2.

Rosowsky, A., J. E. Wright, C. Ginty, J. Uren. 960.

3.

Tansik, R.L., D.R. Averett, B. Roth, S.J. Baccanari. 1984. J. Biol. Chem. 259, 12299.

4.

Kumar, A.A., J.H. Mangum, Biol. Chem. 256, 8970.

5.

Matthews, D.A., R.A. Alden, J.T. Bolin, D.J. Filman, S.T. Freer, R. Hamlin, W.G.J. Hoi, R.L. Kisliuk, E.J. Pastore, L.T. Plante, N. Xuong, J. Kraut. 1978. J. Biol. Chem. 253, 6946.

6.

Volz, K.W., D.A. Kaufman, J. Kraut.

D.T.

J. Biol. Chem. 260, 1465.

Blankenship,

1982. Paterson,

J. Med. Chem. 25, D.

Stone,

J.H. Freisheim.

Matthews, R.A. Alden, S.T. Freer, 1982. J. Biol. Chem. 257, 2528.

C.

1981.

Hansch,

D.P. J.

B.T.

ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE ISOLATED AS A FOLATE COMPLEX

S.S. Joyner and D.P. Baccanari The Wellcome Research Laboratories, Research Triangle Park, NC

27709

Introduction Multiple forms of dihydrofolate reductase (DHFR, EC 1.5.1.3) can be caused by a variety of factors.

For example, the two isozymes of E. coli RT 500 DHFR

(called form 1 and form 2) differ in a single amino acid (1).

Another type

of multiplicity is seen with the chicken liver (2), Lactobacillus casei (3) and L1210 enzymes (4).

These reductases can be isolated as both the free

enzyme and as a naturally occurring, enzyme-NADPH binary complex.

The present

study shows that E. coli RT 500 DHFR exists in multiple enzyme-folate complexes in vivo.

Results E. coli RT 500 DHFR was purified by a combination of gel filtration and hydrophobic chromatography on hexylamine agarose.

The hydrophobic matrix had a

high capacity for the enzyme (>77 units/ml), and elution was easily achieved with a KC1 gradient.

However, under these conditions, three peaks of enzymic

activity were observed (for example, see Fig. 1).

The first had the electro-

phoretic mobility and kinetic properties of the form 2

coli DHFR isozyme,

whereas enzyme from both the remaining peaks was similar to the form 1 isozyme. Although this multiplicity could have been caused by a variety of factors, one interpretation is that the form 1 enzyme was being isolated as both a free enzyme and an enzyme-ligand complex.

This was tested further.

E. coli RT 500 was grown in the presence of [ l 4 C]p -aminobenzoic acid to determine whether or not folates synthesized in vivo were associated with the enzyme.

Cells were harvested, lysed, subjected to ammonium sulfate fractiona-

tion, and applied to an AcA 54 gel filtration column.

Although proteins

eluted throughout the total fractionation range of the column, only two peaks of radioactivity were observed.

One co-eluted with the DHFR activity, and

Chemistry a n d Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

812 the other eluted in the total volume of the column.

Both of these fractions

were shown to contain biologically active folates by their ability to support the growth of L_¡_ case i. The DHFR pool from the gel filtration step was loaded onto a hexylamine agarose column and eluted with KC1. observed (Fig. 1, left).

Three peaks of enzymic activity were

The first had a high pH 5 to pH 7 enzymic activity

ratio, which is characteristic of form 2 RT 500 DHFR (5).

The other two

peaks had higher enzymic activities at pH 7 than at pH 5, which is characteristic of the form 1 isozyme.

Radioactivity was associated only with the

form 2 peak and the first form 1 peak, and of these, only the form 2 peak was capable of supporting the growth of L^ casei (Fig. 1, right).

However,

folates are isolated from E\_ coli (and most other organisms) as polyglutamates containing up to 7 additional glutamate residues (6), yet L c a s e i can only utilize pteroyl mono-, di- or triglutamates.

When the hexylamine agarose

column fractions were treated with hog kidney conjugase to hydrolyze folylpolyglutamates, the radioactive form 1 enzyme peak then supported the growth of U

casei (Fig. 1, right).

Figure 1. Hydrophobic chromatography of DHFR isolated from E\ coli grown in the presence of [ ^C]p-aminobenzoic acid. 30 units of DHFR (specific activity = 41 U/mg) from an AcA 54 gel filtration column were applied to a 10 ml hexylamine agarose column. Left panel: 1 ml fractions were assayed both in 0.1 M imidazole-Cl pH 7 and 0.1 M succinate-Tris pH 5. Right panel: The same fractions were assayed for their 14C radioactivity [ ] and ability to support the growth of casei with [ ] or without [ ] pretreatment with conjugates. The arrows indicate the locations of the three peaks.

813 The three peaks from the hexylamine column were subjected to analytical Polyacrylamide electrophoresis, and each fraction was found to be free of nonreductase proteins (Fig. 2). small amount of form 1.

The first peak was predominately form 2 with a

Peak 2 contained a mixture of the two isozymes, and

the third peak was pure form 1.

Folates (measured by their radioactivity)

dissociated from both isozymes during electrophoresis and co-migrated with the bromophenol blue front.

RELATIVE MOBILITY Figure 2. Polyacrylamide electrophoreses of hexylamine-agarose purified DHFR. Peak 1, left; peak 2, middle; peak 3, right. Gels were stained for protein and scanned at 600 nm. The origins and bromophenol blue fronts are marked.

Discussion The biological significance of the DHFR-folate association is not known, but its existence may be related to the intracellular enzyme concentration. Silhavy et al. (7) have shown that ligands do not readily diffuse from proteins when the concentration of protein is much greater than its K 95% pure.

is not a multimer

and kinetic properties

for this enzyme (7).

The enzyme exhibits

as reported

by others

a Mr

(7).

=

The

are quite different from those reported

Antibodies to the human DHFR bound to the

by

plant

TABLE I. PURIFICATION OF D1HYDR0FQLATE REDUCTASE FROM SOYBEAN SEEDLINGS VOLUME STEP

TOTAL PROTEIN

(ML)

1) POST PROTAMINE

3000

"(KG)

SPECIFIC ACTIVITY

RECOVERY

*(UMOLES/MIH)

TOTAL ACTIVITY

(UMOLES/HIN/HG)

(I)

(10)

0.0005

22.000

2)

MTX-SEPHAR0SE

0.076

3)

ULTR0GEL AcA 51

0.25

4)

BLUE SEPHAROSE

0.053

30 56.6

30

* ONE UNIT OF ACTIVITY IS DEFINED AS THAT AMOUNT OF ENZYME WHICH CATALYZES THE CONVERSION OF 1 UMOLE OF DIHYDROFOLATE TO TETRAHYDROFOLATE PER MINUTE AT pH 7.5 AND AT 45°C. "

PROTEIN IS ESTIMATED BY THE B10RAD PROCEDURE USING BOVINE SERUM ALBUMIN AS A STANDARD.

enzyme on Western blots and cross-reacted significantly (about 70 percent) in immunoassays

indicating some sequence homology between the two enzymes.

The

Km values of NADPH and FAH2 were determined to be 21 yM and 15 yM, respectively.

These values are 5-10 fold higher than those reported for other DHFRs

suggesting

lower

affinity

for

FAH2

and

NADPH

[Table

II].

It

was

also

surprising to find that FA which has a high affinity for the vertebrate DHFRs, had

very

little

affinity for plant (I50

=

130

ferences at site.

TABLE II. KINETIC PROPERTIES OF DHFRS

enzyme

indicating mode

the pM) dif-

in

its

of

binding

the

active

The

soy-

KM (M x IO6) SOURCE

MR

-,

(x 10"3)

FAH?

NADPH

pH

SPF AC1

OPTIMUM

EFFECT O F KCL

EFFECT O F MERCURIAL

SOYBEAN

22

21

15

7.4

15 (22°C)

INHIBITION

NO EFFECT

L. CASE I

18.3

0.36

0.78

6.5

12 (30°C)

ACTIVATION

NO EFFECT

HUMAN

21

0.04

0.25

15 (22°C)

ACTIVATION

ACTIVATION

4; 7.3-8.3

817 bean DHFR exhibits

a single

have two pH optima (8).

pH optimum, unlike other eukaryotic DHFRs which

Human DHFR is activated 1-2 fold in the presence of

0.1 -1.0 M KC1 (8), but KC1 inhibited the soybean enzyme ( I 5 0 = 0.8 M).

Also,

the plant enzyme, unlike human DHFR, showed no activation upon treatment with organomercurials cysteines

either

present

may

in

the

presence

therefore

be

or

absence

inaccessible

of

to

NADPH.

The

modification

two

or,

if

modified, cause no perturbation of the active site.

table

in.

'50/150(methotrexate)

inhibition of various » F R s by wiTiFOLATCs

T2

3

„

soybean

? Y methotrexate

i

*

33300

TRIMETHOPRIM

4Q700

triuetrexate

tJOQOOO

„

900

-,

against

y

"2 NHj ' 0 H J^grs"-»-©-^" „„

8 4

„

3 0 0 0 0 0

Q 5

m e

the

compared

bacterial

and

human

DHFRs

III). enzyme,

The plant like the other

enzymes,

0

10.6

(Table

showed

stoichiometric

a

26

binding to the plant 3 1" enzyme was ca. 37-fold

COOH

weaker

COOM

„ o

„

J ^ ^ i ^ ^ W

prim 123461

176700

1

56

6-fold the

y^

U T W S t S s * *

1 2 7 7 5 5

j^^SW^z-jO^Q)-^ ,20921

53.3

3.5

6

500

24

32

J"1 oR

3 2

l5Q=AM0UNT OF INHIBITOR NECESSARY TO ACHEIVE FIFTY PERCENT ENZYME INHIBITION compounds

were

potent

inhibitors

while

was

greater

than

DHFR.

restingly, ate,

which

potent

ally

trimetho-

binding human

human 128750

quinazoline

inhibi-

tion by MTX with a Kd < 10" 1 0 M. Pyrimethamine

H365

T!

and

were

H

rgjori^'^x^A00"3

nh2

inhibitors

tested e n z

1

^ pyrimethamine

1

"

7299% pure

was 15-20 U/mg p r o t e i n .

amino a c i d sequence a n a l y s i s o f 50 % of

in the crude

The s p e c . a c t i v i t y

confirmed

but r e v e a l e d t h a t

approx.

M15 c o n t a i n e d an a d d i t i o n a l

The two forms c o u l d not be

separated. Broad pH-optima were o b s e r v e d a t

5.5 and 8 . 0 ,

whereas pH optima

f o r mouse S-180 DHFR were a t pH 4 . 5 and 8 . 0 . K i n e t i c f o r the s u b s t r a t e and NADPH were v e r y s i m i l a r bovine-,

mouse-,

and rat-DHFR

between human-, b o v i n e - , testing

(Table

(Table

2,

t o those

1 ) . Small

of

differences

mouse- and rat-DHFR were r e v e a l e d by

t h e enzyme w i t h s e v e r a l

inhibitors,

constants

structurally

different

Fig.3).

During c h r o m a t o f o c u s i n g o r chromatography on a B i o r e x - 9

column,

s t a b i l i t y problems were e n c o u n t e r e d w i t h t h e h-DHFR i n

contrast

t o t h e mouse S-180 enzyme. The p i PAG-plates

(LKB,

pH 3 . 5 - 9 . 5 )

focusing process,

could not be d e t e r m i n e d

due t o p r e c i p i t a t i o n d u r i n g

p r o b a b l y caused by removal of

enzyme was more s t a b l e i n phosphate b u f f e r , Tris-HCl,

i n t h e p r e s e n c e of

4000 as t h e p r e c i p i t a t i n g They b o t h d i f f r a c t meters a r e as Form A :

bound f o l a t e .

pH 7 . 0 ,

than

The

in

pH 7 . 8 .

The pure h-DHFR, o b t a i n e d lized

on the

from t h e a f f i n i t y

step,

was

crystal-

MTX and NADPH u s i n g p o l y e t h y l e n e agent.

Two c r y s t a l

forms were

t o h i g h r e s o l u t i o n and t h e i r

glycol

obtained.

unit c e l l

para-

follows:

s p a c e group P3^12 w i t h a = b = 3 8 . 5 A and c = 2 2 6 . 8 A

Form B: space g r o u p C2 w i t h a = 65.8 A,

b = 4 0 . 1 A,

and B = 1 1 0 . l o . X-ray s t r u c t u r a l

studies are in

progress.

c = 76.3 A

842 Table 1:

Comparison of K m -values for substrate and NADPH Km

Substrate

Human

(^M) for DHFR from

Bovine

Mouse S-180

Rat

Dihydrofolate

0.72

1.2

0.57

0.79

NADPH

2.5

1.0

1.9

1.8

Table 2

Inhibition constants of several inhibitors Ki (nM) for DHFR from

Inhibitor

Human

Bovine

Mouse S-180

0. 017

0.027

Compound 1

7.33

2 . 99

18 . 1

Compound 2

5.04

0.33

2 .6

Methotrexate

Compound 3

487

418

3 2

NH2

y ° 'm

t y j V v 1

kA^J

0 . 033

NH;

Hs n

576 • 0CH:

h

2

3

Figure 3: Structures of some inhibitors tested (Table 2)

References 1. Matthews, D .A . , J.F.Bolin, J.M.Burridge, D.J.Filman, K.W.Volz, B.T.Kaufman, C.R.Beddell, J.N .Champness, D.K.Stammers, J.Kraut. 1985. J.Biol.Chem. 260, 381-391 2. Kuyper, L.F., B.Roth, D.P.Baccanari, R.Ferone, C.R.Beddell, J .N.Champness, D.K.Stammers, J.G.Dann, F.E.Norrington, D.J.Baker, P.J.Goodford. 1985. J.Med.Chem. 28, 303-311 3. Kompis, J., R.L.Then. 1984. Eur.J.Med.Chem. 19, 529-534 4. Masters, J.N., G.Attardi. 1983. Gene 21, 59-63 5. Stueber, D., I.Ibrahimi, D.Cutler, B.Dobberstein, H.Bujard. 1984. EMBO J. 3, 3143-3148

LEVELS OF FOLATES AND METHOTREXATE POLYGLUTAMATE FORMATION IN CHINESE HAMSTER OVARY CELLS LACKING DIHYDROFOLATE REDUCTASE

P. Joannon, H. Goldberg, V.M. Whitehead, D.S. Rosenblatt, M.J. Vuchich, D. Beaulieu McGill University-Montreal Children's Hospital Research Institute, Montreal, Canada, H3H 1P3

Introduction Accumulation of methotrexate polyglutamates (MTXPGs), particularly those with more than 3 glutamyl residues, appears to contribute both quantitatively and qualitatively to MTX cytotoxicity (1).

We have found that mutant (DUK) Chinese

hamster ovary (CHO) cells which lack dihydrofolate reductase (DHFR) (2) accumulate higher levels of MTXPGs and longer chain-length MTXPGs than do control (UTC) CHO cells (3).

We

report levels of folates in these two cells as well as evidence that differences in folates do not explain the differences in MTXPG formation.

Methods Both DUK and UTC were grown in monolayer culture in modified Ham's F12 medium which contains glycine, hypoxanthine, thymidine and 2.3 mM folic acid together with 10% fetal calf serum (FCS). Heated cell extracts were treated with hog kidney tf-glutamyl hydrolases.

Folates were assayed by Dr. B.A. Cooper using

differential microbiological assay (4).

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

844

UTC were folate depleted by growing them in folate-free Eagle's minimal essential medium (MEM) with the addition of 10% undialysed FCS.

After 7 days, doubling times increased from

about 20 to 70 hours. /uM folic acid.

Controls were grown in MEM containing 2.3

Starting on days 8,9 and 10, cells were

incubated with 1.0 /uM [ 3 H]MTX (Moravek Co.) for 24 h.

After

incubation, cell extracts were prepared directly (total MTX) or after 1 h incubation in MTX-free medium (non-exchangeable MTX) and analysed by HPLC (5).

Results Levels of total folates in UTC measured by L. casei were higher than in DUK (Table 1).

About half the folates were active for

growth of S. fecalis and P. cerevisiae, consistent with the presence of reduced folates such as 5-CHO-H4folate, lO-CHOH4folate, 5,10-CH2~H4folate and I^folate.

The remaining

folate which was detected by L. casei only was 5-CH3~H4folate. In DUK, all the folate was active for growth of L. casei and S. fecalis, but not for P. cerevisiae identifying it as folic acid. Since the predominant folate supplied was folic acid, this finding provides additional evidence of the absence of DHFR in these cells.

The complete absence of reduced folates from DUK suggests

that they do not utilize or retain reduced folates present in the FCS.

To determine whether reduced MTXPG formation in UTC was due to their increased level of reduced folates compared to DUK, UTC were depleted of folates by prolonged growth in low-folate medium and MTXPG formation studied (Table 2).

No increase in levels of total

845 TABLE 1 Cell line

LEVELS OF TOTAL FOLATES IN UTC AND DUK CELLS n

Lactobacillus casei

Pediococcus cerevisiae

Streptococcus fecalis

nmol/g protein (mean ± 1SD) UTC

8

40.2+7.2

DUK

8

6.1+2.3

TABLE 2

Folate status

16.5+3.8 0.0

n

control

lh Efflux

intracellular MTX and MTXPGs - nmol/g (% total)

12.5

control

deficient

7.4+2.8

EFFECT OF FOLATE DEPLETION ON TOTAL AND NON-EXCHANGEABLE MTX AND MTXPGs UTC

Total

deficient

26.9+5.2

9.7

3

3

12.4

8.3

MTX

MTX

MTX

MTX

G1U2

G1U3

GIU4

TOTAL MTXPGs

7.4

2.0

2.8

0.4

5.1

(59)

(16)

(22)

(3)

(41)

4.0

2.1

3.1

0.5

5.7

(41)

(22)

(32)

(5)

(59)

8.2

1.8

2.2

0.2

4.2

(66)

(14)

(18)

(2)

(34]

4.0

1.7

2.4

0.2

4.3

(48)

(21)

(29)

(2)

(52)

846

and non-exchangeable MTX, in proportion of total MTXPGs or in the predominant chain-length of MTXPGs was found in folate-depleted UTC compared to controls.

Prolongation of the

doubling time of UTC after growth in low folate medium for 7 days indicated that they were folate deficient.

These findings

support the hypothesis that the longer chain-length MTXPGs formed in DUK cells compared to UTC is due to differences in the level of DHFR.

Binding of MTXPGs to DHFR as they are formed

interferes with their further elongation (3).

References 1.

Chabner, B.A., C.J. Allegra, G.A. Curt, N.J. Clendennin, J. Baram, S. Koizumi, J.C. Drake, J. Jolivet. 1985. J. Clin. Invest. 76, 907.

2.

Urlaub, G., L.A. Chasin. 1980. Proc. Natl. Acad. Sei. 77, 4216.

3.

Joannon, P., V.M. Whitehead, D.S. Rosenblatt, M.-J. Vuchich, D. Beaulieu. 1986. Proc. Amer. Assn. Cancer Res. 21_, 254.

4.

Cooper, B.A., E. Jonas. 1973. J. Clin. Path. 26, 963.

5.

Whitehead, V.M., D.S. Rosenblatt. 1985. In: Proceedings of the Second Workshop on Folyl and Anti-folyl Polyglutamates (I.D. Goldman, ed.). Praeger Scientific, p. 214.

METHOTREXATE

IN A D J U V A N T

ARTHRITIS

John Galivan, Mary-Catherine

Rehder

W a d s w o r t h C e n t e r for L a b o r a t o r i e s a n d R e s e a r c h , D e p a r t m e n t of H e a l t h , A l b a n y , N e w York 1 2 2 0 1

Suresh

N e w York

State

Kerwar

D e p a r t m e n t of I n f l a m m a t i o n a n d I m m u n o l o g y , M e d i c a l R e s e a r c h Division, American Cyanimide Company, Lederle Laboratories, Pearl River, New York 10965

Introduction Numerous clinical v a l i d i t y of

studies

in r e c e n t y e a r s h a v e d e m o n s t r a t e d

low dose m e t h o t r e x a t e

rheumatoid arthritis

(1-5).

in the a m e l i o r a t i o n

Because

little

the m e c h a n i s m o f a c t i o n of m e t h o t r e x a t e we have

i n i t i a t e d a s t u d y of

on animal models arthritis that used

this

We have shown that

to d o s e s of m e t h o t r e x a t e

arthritis

antimetabolite adjuvant

(6) a n d s t r e p t o c o c c a l c e l l w a l l a r t h r i t i s

both respond

concerning

in r h e u m a t o i d

the e f f e c t of

for a r t h r i t i s .

is k n o w n

the

of

(7) in

that are equivalent

in t h e t r e a t m e n t of r h e u m a t o i d a r t h r i t i s

rats to

in m a n .

Further studies have been u n d e r t a k e n with the a d j u v a n t model determine

a) t h e i m p o r t a n c e

achieving

a therapeutic

of the t i m i n g

response

requirements

of the a n t i f o l a t e .

are reported

here.

Results and

the

treatment

in

structural

T h e r e s u l t s of t h e s e

low d o s e m e t h o t r e x a t e

inflammation associated with adjuvant

w h e n g i v e n a t a d o s a g e of to d e t e r m i n e

of d r u g

b) the

studies

Discussion

Our prior study has shown that alleviate

and

to

15 )jg o n a b i w e e k l y

if e f f e c t i v e n e s s

basis.

can arthritis In o r d e r

c o u l d be r e t a i n e d by r e d u c i n g

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

the

848 Table

I:

E f f e c t s of A n t i f o l a t e s o n Paw I n f l a m m a t i o n with Adjuvant

Agent

Dose

Arthritic

Index

12.0 + 0

None Methotrexate

0.0 + 0.0

15 ug

Aminopterin Bakers

Associated

Arthritis

antifol

CB3717 Trimétrexate

15 u g *

6.9 + 1.1

120 tig*

4.0 + 4.0 0.0 + 0.0

30 yg 600 yg 300 yg

10

1.6

11.7 + 0.5

600 yg 300 yg

Metoprine

+

12.0 + 0 10.8 + 2.2

The i n d u c t i o n of a d j u v a n t a r t h i t i s and scoring of

inflammation

(arthritic index) were c o n d u c t e d as p r e v i o u s l y d e s c r i b e d R e s u l t s are e x p r e s s e d as the m e a n +_ S.D. (N=4). at the i n d i c a t e d dose twice w e e k l y (*) w h i c h w a s o n c e

(6).

Drug was g i v e n

i.p. e x c e p t as indicated by

weekly.

frequency of d o s a g e m e t h o t r e x a t e w a s a d m i n s t e r e d o n c e w e e k l y (Table 1).

Restriction

in the 15 yg d o s a g e o n c e weekly

a severe r e d u c t i o n in t h e r a p e u t i c e f f e c t .

A n increase

caused in the

o n c e w e e k l y d o s a g e to 120 yg did not s i g n i f i c a n t l y e n h a n c e r e d u c t i o n in i n f l a m m a t i o n .

These results s u g g e s t e d

importance of the timing of m e t h o t r e x a t e suppressing

treatment

in

inflammation.

In o r d e r to test the s t r u c t u r a l

s p e c i f i c i t y of m e t h o t r e x a t e

the a d j u v a n t m o d e l , s e v e r a l a n t i f o l a t e s were e v a l u a t e d . n u m b e r of d i f f e r e n t d i h y d r o f o l a t e widely different structure

reductase

than m e t h o t r e x a t e .

i n e f f e c t i v e u n d e r these c o n d i t i o n s .

was

Only a m i n o p t e r i n , showed

which

significant

H o w e v e r , s y m p t o m s of toxicity

o b s e r v e d e v e n at the d o s e s used h e r e .

and

primarily

s y n t h a s e , CB3717 or p r o p a r y l q u i n a z o l i n e

r e d u c t i o n in i n f l a m m a t i o n .

A

10 to 20 fold h i g h e r

The a n t i f o l a t e w h i c h i n h i b i t s

s t r u c t u r a l l y h i g h l y similar to m e t h o t r e x a t e

on

i n h i b i t o r s of

(Bakers a n t i f o l , t r i m e t r e x a t e ,

m e t o p r i n e ) h a d no e f f e c t at c o n c e n t r a t i o n s thymidylate

the

the

Further studies

are

were

849 needed to determine if lower, non-toxic doses of aminopterin can be used in place of methotrexate. These results demonstrate that anti-inflammatory activity

in

adjuvant arthritis is exhibited only by analogs that have a structure highly similar to methotrexate. It is not certain this time if dose modification or other structural

alterations

may enhance the activity of the compounds shown to be inactive here. It is difficult to make direct comparisons between animal models of arthritis and rheumatoid arthritis since differences between the human and animal pathology have been established (8). However the similarity in dose response to methotrexate between the human disease and two animal models suggest that useful information concerning the therapeutic efficacy and mechanism of action of antifolates in the treatment of arthritis may be obtained

in the model systems.

Acknowledgement

This study was supported by NIH Grants CA25933 and CA34314.

References 1.

Wilkens, R.F., M.A. Watson. 1982. 314-321

J. Lab. Clin. Med. 100,

2.

Weinblatt, M.E., J.S. Colby, D. A. Fox et. al. 1982. N. Engl J. Med. l\2> 818-822.

3.

Steinsson, K., A. Weinstein, M. Abeles. 1982. J. Rheumatol. 8, 860-866.

4.

Russell, A.S., C. Watts, R. Thompson, et al. 1984. Arthritis Rheum. 21_, 559.

5.

Williams, H.J., R.F. Wilkens, C.D. Samuelson, Jr. et al. 1985. Arthritis Rheum. 28^, 721-729.

6.

Welles, W.L., J. Silkworwth, A.L. Oronsky, S.S. Kerwar, J. Galivan. 1985 . J. Rheumatol. 904-906.

7.

Ridge, S.C., N. Rath, J. Galivan, J. Zabrisbe, A.L.

Oronsky, S.S. Kerwar: J. Rheumatol., in press. Decker, J. L., Mulone, D. G., Haraqui, B. et al. Intern. Med. 101, 810-824, 1984

Ann.

COMPUTER GRAPHIC MODELING IN DRUG DESIGN:

CONFORMATIONAL ANALYSIS AND ACTIVE-

SITE MODELING OF LIPOPHILIC DIAMINOPYRIMIDINES

Vivian Cody Medical Foundation of Buffalo, Inc., 73 High St., Buffalo, New York

14203 USA.

Introduction The enzyme dihydrofolate reductase (DHFR), a necessary component for all cell growth, is strongly and specifically inhibited by certain substrate analogues with binding affinities so great that they are not readily displaced by the natural folic acid substrates.

These antifolates have been the focus of chemo-

therapy for infectious and neoplastic diseases because of their ability to interfere in the synthesis of purine and pyrimidine nucleotide precursors of DNA and RNA (1,2).

Lipophilic diaminopyrimidines are a class of drugs that act as

inhibitors of DHFR and which show striking differences in their inhibitory activity with only small changes in their structure (1,2). Moreover, among 5-adamantyl 6-substituted antifolates, there is a further sharp increase in cytotoxic activity as the 6-substituent increases from hydrogen to methyl to ethyl, but drops again at propyl (3).

These lipophilic antifolates have the advantage of

a more extensive cellular uptake, with rates about 10,000 times more rapid than methotrexate (MIX), the most effective and most widely used anticancer agent, and show strong cytotoxic activity in cultured cells (4). In order to delineate the structural, conformational and electronic properties which are important for species specificity and selectivity, X-ray crystallographic analyses were carried out on a series of lipophilic antifolates (5,6) and computer graphic modeling of the binding interactions of these antifolates within the active site of chicken liver DHFR were investigated (7,8).

Structural Results Crystallographic analyses of 5-(1-adamantyl) diaminopyrimidine antifolates (Fig. 1) reveals that within the series of 6-substituted analogues (DAHP, DAMP, DAEP, DAPP), the pyrimidine ring in each structure is distorted from planarity

Chemistry and Biology of Pteridines 1986 © 1986 Walter d e Gruyter & Co., Berlin • New York - Printed in Germany

852

Fig. 1.

Structure of a) DAHP, b) DAMP, c) DAEP, d) DAPP, e) DCXMP, and f) DTMP.

with its substituents, with the exception of DAHP, which is planar.

As a re-

sult of the steric strain placed upon the system by the close intramolecular interactions of the adamantyl hydrogen atoms and those of the 4,6-substituents, these molecules have a bowed shape (6-8).

This strain causes the pyrimidine

ring to become a flattened boat with C(2) and C(5) below the N(l), N(3), C(4), C(6) plane.

As illustrated (Table I), these distortions cause the exocyclic

groups, to be significantly displaced from the pyrimidine plane. positionaliy disordered in both DAEP and DTMP.

Atom C(61) is

Since the high binding affinity

of antifolates for the enzyme active site is assumed to involve strong multiple hydrogen bonding interactions (9), these pyrimidine ring distortions can influence the placement of the diaraino groups and affect the strength and directionality of their hydrogen bonds in the enzyme active site. Table I:

Deviations of Substituents from the Pyrimidine Nl, N3, C4, C6 Plane.

Structure DAHP DAMP DAEP1 DAEP2 DAPP DCXMP DTMP1 DTMP2

N(2)

N(4)

C(7)

0,.015 -U,.233 -0,.254 -0,.254 -0,.291 -0..052 0..040 0..040

-0..040 0..238 0..241 0,.241 0,.372 0..003 -0,.093 -0,.093

0 .036 -0 .419 -0 .461 -0 .461 -0 .470 0 .061 0 .240 0 .240

C (61) 0..031 0 .055 0 .418 -0 .405 0 .180 -0 .020 -0 .217 0 .280

C(2) 0 . 009 -0 .088 -0 .087 -0 .087 -0 .132 -0 .008 0 .019 0 .019

C(5) 0 .024 -0 .121 -0 .122 -0 .122 -0 .160 0 .022 0 .084 0 .084

As illustrated (Fig. 2), the deviations of C(7) and N(4) in the adamantyl antifolates (DAEP, DAMP, DAPP) are significantly different from the more planar structures.

These displacements are correlated in pairs:

N(4); C(61), C(7), with those of DAPP the most variable.

C(2), C(5); N(2),

853

N(2) -•- N(4) * C(7) > h; > -o- C(61) Moo

a

v

C(2) C(5)

DAEP1DAEP2 DAMP DAPP DAHPDCXnFOTMP 1DTMP2 ANALOGUE

Fig. 2 Activity correlation with substituent displacement from pyrimidine plane. Crystallographic data delineating the DHFR enzyme structure and inhibitor/cofactor complexes are available from two bacterial and one avian species (10,11), and show that the active site is located within a 15$ cavity cutting across one face of the enzyme.

Structural data show that DAMP also binds to chicken liver

DHFR in a similar manner (11).

Modeling the interactions of DAMP in the DHFR

active site shows that the adamantyl ring fits tightly into the hydrophobic pocket near that occupied by the p-aminobenzoyl ring of MTX, and that the 6methyl is in a hydrophobic pocket surrounded by the residues Trp-24, Glu-30 and Tyr-31. Investigation of the 6-ethyl environment of DAEP shows that, while there is a reasonable fit, a 110° rotation of the ethyl side chain moves it into a more suitable pocket (6).

A similar comparison of the 6-propyl side chain indicates

that it also fits into this space.

In its observed orientation, the propyl

side chain atom C(63) is 2.38X from the hydroxyl oxygen of Tvr-31. o

A 60° ro-

tation of the side chain increases this distance to 3.64A (8). These observations suggest that other 6-substituted antifolates can be proposed which could take advantage of specific interactions with these residues.

Ac-

cordingly, computer generated models were made for other 6-substituted 5-adamantyl antifolates and their fit tested in this pocket. toxy group

For example, a 6-ace-

has an 0...0 contact distance to Tyr-31 of 3.3oX, favorable for

hydrogen bonding.

854 Summary The results of these structural studies show that the pyrimidine ring and its substituents become more distorted from planarity as the size of the 6-substituent increases and that there is a correlation between the activity of these adamantyl antifolates and the displacement of C(7) and N(4) from the leastsquares plane through Nl, N3, C4, C6.

These data also indicate that other pa-

rameters are of importance to the binding affinity of these lipophilic antifolates which could better explain the activity of DAEP. Acknowle dgement s This research was supported in part by NCI CA-34714, American Cancer Society Faculty Research Award, FRA-287, and the Buffalo Foundation. References 1.

Blakely, R.L. 1984. In: Folates and Pterins (R.L. Blakely and S.J. Benkovic, eds.). John Wiley f, Sons, New York, Vol. 1, p. 191.

2.

Zakrzewski, S.F., Dave, C. § Rosen, F. 1978. J. Natl. Cancer Inst. 60, 1029.

3.

Jonak, J.P., Zakrzewski, S.F. § Mead, L.H. 1971. J. Med. Chem. 14, 408; 1972. 15, 662. ~~

4.

Greco, W.R. § Hakala, M.T. 1982. Mol. Pharmacol. 18, S21.

5.

Cody, V. § Zakrzewski, S.F. 1982. J. Med. Chem. 25, 427.

6.

Cody, V., Welsh, W.J., Optiz, S. q Zakrzewski, S.F. 1984. In: QSAR in Design of Bioactive Compounds (Kuchar, M., ed.). J.R. Prous Science, Barcelona, Spain, p. 241.

7.

Cody, V. 1984. In: Molecular Basis of Cancer, Part B: Macromolecular Recognition, Chemotherapy and Immunology (Rein, R., ed.). Alan R. Liss, Inc., New York, p. 275.

8.

Cody, V. 1986. J. Mol. Graphics 4, 69.

9.

Zakrzewski, S.F. 1963. J. Biol. Chem. 238, 1,485; 4002.

10.

Bolin, J.T., Filman, D.J., Matthews, D.A., Hamlin, R.C. § Kraut, J. 1982. J. Biol. Chem. 257, 13650.

11.

Matthews, D.A., Bolin, J.T., Burridge, J.M., Filman, D.J., Volz, K.W., Kaufman, B.T., Beddell, C.R., Ch;unpness, J.N., Stammers, D.K. § Kraut, J. 1985. J. Biol. Chem. 260, 381, 392.

EFFECT OF ANTIFOLATES 10-METHYL- AND 10-ETHYL-10-DEAZA-AMIN0PTERIN ON A HUMAN BREAST CANCER CELL LINE

Frederika Mandelbaum-Shavit

Department of Bacteriology, Jerusalem, Israel.

Hebrew University-Hadassah

Medical

School,

Introduction

New folate analogs of the 10-deaza-aminopterin

series were shown during the

recent years to be less toxic to normal tissues and more effective against several

tumors of murine and human origin than methotrexate (MTX) (1-4).

Analogs with methyl or ethyl group at the 10 position of 10-deaza-aminopterin were more effective than MTX in reduction of tumor and increasing s u r v i v a l time of mice with L1210 or S180 ascites tumor and studies like E0771 mammary adenocarcinoma,

with solid

tumors

T241 fibrosarcoma and others proved

also

that 10- ethyl-10-deazaminopterin (lO-Et-lOdAm) was 5-fold more effective than MTX in retarding tumor growth (2). Experiments ¿n vitro

supported the above

results showing that 10-methyl- and 10-ethyl- derivatives are more potent inhibitors of various cells grown in culture, than MTX (3). We now present data on cytotoxicity

of

lO-Et-lOdAm and the 10- methyl-

derivative (lO-Me-lOdAm) as compared to that of MTX in MCF-7 cells, a human breast cancer cell line.

Since

5-methyltetrahydrofolate (5-CH3-H^PteGlu) is

a normally circulating form of folate (5), it appeared also of importance to study transport of this derivative vs MTX and the 10-deaza analogs.

Results

Cytotoxicity of Methotrexate, 10-Methyl- and 10- Ethyl-10-deaza-aminopterin in MCF-7 cells. The results depicted in Fig. 1 show that upon continuous exposure to various

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

856 concentrations of the f o l a t e analogs,

lO-me-lOdAm and 10-Et-dAm were 3.6 and

3 . 2 - f o l d r e s p e c t i v e l y more c y t o t o x i c than MTX (comparing required for 50% inhibition,

concentrations

IC^Q)

Figure 1. Inhibition of growth of MCF-7 c e l l s by antifolates. Chemicals: 10Me-lOdAm and lO-Et-lOdAm were synthesized by Dr. Joseph I. DeGraw, Department of Pharmaceutical Chemistry, Stanford Research Inst., Menlo Park, California (1) and kindly provided by P r o f . Roy L. K i s l i u k , Dept. of Biochemistry and Pharmacology, Tufts University School of Medicine, Boston, Massachusetts. The analogs were p u r i f i e d by chromatography and the concentrations determined s p e c t r o p h o t o m e t r i c a l l y (1). C e l l growth: MCF-7 c e l l s were c u l t i v a t e d in Earle's.based minimal e s s e n t i a l medium supplemented with 10% f e t a l bovine serum (Grand Island B i o l o g i c a l Co., NY), 0.2 U/ml of i n s u l i n , non e s s e n t i a l amino acids, 1 mM sodium pyruvate, 2 mM glutamine, 100 U/ml of p e n i c i l l i n and 100 ug/ml of streptomycin. C e l l s were subcultured following resuspension in a RDB s o l u t i o n s ( a novel agent of plant o r i g i n f o r dispersing monolayer culture, product of Israel Inst, for Biological Research, Ness Ziona), diluted in medium and 2 ml of the c e l l suspension (about 10 c e l l s were plated i n t o 35x10 mm t i s s u e c u l t u r e dishes. A f t e r incubation f o r about 40 hr in a humidified incubator under an atmosphere of air with 5% COn at 37°C, to allow enter the exponential growth phase, the medium was removed and the drugs were added in 2 ml of fresh medium. The cultures were reincubated and after 3 days resuspended and counted. Symbols: MTX (t); lO-Me-lOdAm (0); lO-Et-lOdAm (*•). Transport of 5- M e t h y l t e t r a h y d r o f o l a t e , Methotrexate, 10- E t h y l - and 10Methyl-10-deaza-aminopterin. In the presence of 2 >iM of S-CHg-H^PteGlu (both stereoisomers) the influx was linear

for

about 8-10 min and a double

accumulation against extra c e l l u l a r typical

reciprocal

plot

of

substrate

concentration (0.2-10 jiM) exhibited

Michaelis-Menten kinetics (not shown). The apparent Km for S-CHg-H^

PteGlu was 3.2+0.28 jiM, e s s e n t i a l l y as the Ki determined with [ 3 H] MTX and the Vmax was 11.6^0.71 nmol/min/g protein. The apparent Km f o r transport of MTX was 7.85+0.61 JJM, a v a l u e c l o s e to that obtained by Schilsky et a l . f o r MTX transport in MCF-7 c e l l s (6),

and the Vmax was 6.50^0.52 nmol/min/g protein.

The Ki values obtained for 10—Et—lOdAm and 10—Me—10-dAm, when measured with

857

[ 3 H]MTX

or

[UC]5-CH3-H4PteGlu

were

lower

by 3 . 3 ' a n d

about

5-fold,

r e s p e c t i v e l y , t h a n t h a t f o r MTX ( T a b l e 1 ) . T h i s i n d i c a t e s t h a t t h e 1 0 - d e a z a analogs are better

s u b s t r a t e s f o r t h e t r a n s p o r t c a r r i e r f o r f o l a t e s than MTX

w i t h a p p a r e n t Ki v a l u e s c l o s e r t o t h a t of t h e n a t u r a l

substrate,

5-CHg-

H 4 PteGlu. T a b l e 1.

T r a n s p o r t of 5 - M e t h y l t e t r a h y d r o f o l a t e and A n t i f o l a t e s i n MCF-7

Cells. Compounds

I n f l u x Ki (uM) A

5-CH 3 -H 4 PteGlu

3.17

MTX

7.90

10-Et-10dAm

2.40

10-Me-10dAm

1.61

+ + + +

B 0.24a

3 .20

0.63

7,.83

0.23

2,.34

0.20

1,.50

+ + + +

0 .16 0 .58 0,.18 0 .14

[ 3 ' , 5', 7- 3 H] MTX, sodium s a l t , sp. a c t . 18 Ci/mmol and 5 - [ 1 4 C ] methylt e t r a h y d r o f o l i c a c i d , s p . a c t . 5 8 . 3 . mCi/mmol, p u r c h a s e d f r o m Amersham, England, were p u r i f i e d and q u a n t i t a t e d by s p e c t r o p h o t o m e t r y (7). Exponential c u l t u r e s a t a l m o s t c o n f l u e n c y i n 3 5 x 1 0 mm d i s h e s were washed w i t h PBS and i n c u b a t e d i n 2 ml of t h e u p t a k e s o l u t i o n ( i n g / L : C a C l ^ - 0 . 2 ; KC1 - 0 . 4 ; MgS0 4 .7H 2 0 - 0 . 2 ; NaCl - 6 . 8 ; NaHC0 3 - 2 . 2 ; NaHnPO^.HjO - 0 . 1 4 , g l u c o s e 0.1%) f o r 1 hr a t 37°C. The s o l u t i o n was removed and t n e l a b e l e d coumpound was added i n 1 ml of f r e s h s o l u t i o n . The c e l l s w e r e i n c u b a t e d a t 37°C. U p t a k e was t e r m i n a t e d by r a p i d c o o l i n g and 3 w a s h e s w i t h i c e - c o l d PBS. The c e l l s w e r e d i g e s t e d i n IN NaOH, n e u t r a l i z e d and c o u n t e d . P a r t of t h e NaOH d i g e s t was used f o r p r o t e i n d e t e r m i n a t i o n (7). I n i t i a l u p t a k e k i n e t i c s were determined i n c e l l s i n c u b a t e d f o r 1 min with v a r i o u s c o n c e n t r a t i o n s of t h e compound examined and Ki v a l u e s were d e r i v e d from d o u b l e r e c i p r o c a l p l o t s of drug a c c u m u l a t i o n v e r s u s c o n c e n t r a t i o n i n t h e absence and p r e s e n c e of t h e competing a n a l o g . A - Ki v a l u e s d e r i v e d from e x p e r i m e n t s measuring i n f l u x of M h ] MTX; B - Ki v a l u e s d e r i v e d from e x p e r i m e n t s measuring i n f l u x of [ C] S-CHg-H^PteGlu; a Mean i S . E . of t h r e e e x p e r i m e n t s in t r i p l i c a t e . I n h i b i t i o n of D i h y d r o f o l a t e Reductase. The v a l u e s d e p i c t e d

in Table 2 a r e derived

from i n h i b i t i o n p r o f i l e s of DHFR

a c t i v i t y o b t a i n e d in t h e p r e s e n c e of v a r i o u s drug c o n c e n t r a t i o n s .

The enzyme

from MCF-7 c e l l s e x h i b i t e d o n l y a s l i g h t l y h i g h e r s e n s i t i v i t y t o t h e 1 0 - d e a z a a m i n o p t e r i n a n a l o g s a s compared t o t h a t a c h i e v e d with MTX.

858 T a b l e 2. I n h i b i t i o n of D i h y d r o f o l a t e R e d u c t a s e by M e t h o t r e x a t e and Other Antifolates Compound

Drug concentration for 50% inhibition (nM) 4..20

+

0 ,25 a

10-Et-10dAm

3..87

+

0 .30

10-Me-10dAm

3..94

+

0 .36

MTX

E x p o n e n t i a l l y growing c e l l s were washed with PBS and the monolayers were suspended in PBS c o n t a i n i n g 0.1% EDTA. F o l l o w i n g c e n t r i f u g a t i o n , the c e l l s were resuspended in 10 mM Tris-HCl, pH 7.5,containing 1'mM EDTA and sonicated. The supernatant obtained a f t e r c e n t r i f u g a t i o n (4°C) at 45,000xg for 45 min was u s e d a s enzyme s o u r c e . DHFR (EC 1 . 5 . 1 . 3 ) a c t i v i t y was a s s a y e d s p e c t r o p h o t o m e t r i c a l l y ( 9 ) . The a s s a y mixture in 1 ml c o n s i s t e d of 100 mM potassium phosphate b u f f e r , pH 6.8, 200 mM KC1, 0.1 mM NADPH and e n z y m e . T h e r e a c t i o n was i n i t i a t e d by a d d i t i o n of 0.08 mM d i h y d r o f o l a t e with 10 mM 2 m e r c a p t o e t h a n o l . The a b s o r b a n c e change a t 340 nm was monitored f o r 4 min a t 30°C. For i n h i b i t i o n a s s a y s v a r i o u s drug c o n c e n t r a t i o n s were added t o the reaction mixture, containing 2 enzyme units (1 unit of a c t i v i t y i s the amount of enzyme reducing 1 nmol of dihydrofolate in 1 min) and incubated for 2 min prior to addition of d i h y d r o f o l a t e . The s p e c i f i c a c t i v i t y of the enzyme used was 4.2 units/mg protein. a

Mean

S.E. of t r i p l i c a t e determination for each drug concentration.

Discussion We have shown t h a t

lO-Et-lOdAm and lO-Me-lOdAm a r e 3.2- and 3.6 - f o l d

r e s p e c t i v e l y more potent growth i n h i b i t o r s for MCF-7 c e l l s than MTX (Fig. 1). These r e s u l t s corroborate with the data of others showing increased a c t i v i t y of the 10-deaza-aminopterin analogs a g a i n s t various tumor c e l l l i n e s , as well as against f o l a t e requiring microorganisms (1-4). The f o l a t e analogs and S-CH^-H^PteGlu were found to enter the MCF-7 c e l l s by the same c a r r i e r mediated mechanism exhibiting a markedly higher a f f i n i t y for lO-Et-lOdAm and lO-Me-lOdAm than f o r MTX. In s t u d i e s of i n h i b i t i o n of DHFR a c t i v i t y the deaza-compounds were only s l i g h t l y more e f f e c t i v e than MTX. In conclusion, the higher c y t o t o x i c i t y of lOEt-lOdAm and lO-Me-lOdAm than that of MTX in MCF-7 c e l l s appears to be r e f l e c t e d mainly by the lower i n f l u x Ki v a l u e s , which i s in agreement with the r e s u l t s obtained with other tumor c e l l l i n e s (1,3,4).

859 Acknowledgment

T h i s r e s e a r c h was supported Association.

in p a r t

by a g r a n t from the I s r a e l

Cancer

The author i s g r a t e f u l to P r o f e s s o r s Roy L. K i s l i u k and Joseph

I. DeGraw for kindly providing the deaza- compounds. References 1.

DeGraw, J . I . , V.H. Brown, H. Tagawa, R.L. K i s l i u k , Y. Gaumont and F.M. S i r o t n a k . 1982. S y n t h e s i s and antitumor a c t i v i t y of 1 0 - a l k y l - 1 0 d e a z a m i n o p t e r i n s . A c o n v e n i e n t s y n t h e s i s of 10-deazaminopterin. J . Med. Chem. 25, 1227-1230.

2.

S i r o t n a k , F.M., J . I . DeGraw, F.A. Schmid, L . J . Goutas New f o l a t e analogs of the 10-deaza-aminopterin s e r i e s . Pharmacol. ^2, 26-30.

3.

S i r o t n a k , F.M., J . I . DeGraw, D.M. Moccio, L.L. Samuels and L . J . Goutas. 1984. New f o l a t e a n a l o g s of the 1 0 - d e a z a - a m i n o p t e r i n s e r i e s . Cancer Chemother. Pharmacol. 12, 18-25.

4.

Moccio, D.M., F.M. S i r o t n a k , L.L. S a m u e l s , T. Ahmed, A. Yagoda, J . I . DeGraw and J.R. P i p e r . 1984. S i m i l a r s p e c i f i c i t y of membrane t r a n s p o r t f o r f o l a t e a n a l o g u e s and t h e i r m e t a b o l i t e s by murine and human tumor c e l l s : A c l i n i c a l l y directed laboratory study. Cancer Res. 44^ 352-257.

5.

H e r b e r t , V., A.R. L a r r a b e e and J.M. Buchanan. 1962. S t u d i e s on the i d e n t i f i c a t i o n of a f o l a t e compound in human serum. J . Clin. I n v e s t . 41, 1134-1138.

6.

S c h i l s k y , R.L., B.D. B a i l e y and B.A. Chabner. 1981. C h a r a c t e r i s t i c s of membrane transport of methotrexate by cultured human breast cancer c e l l s . Biochem. Pharmacol. 30, 1537-1542.

7.

Mandelbaum-Shavit, F. and N. Grossowicz. 1970. Transport of f o l i n a t e and r e l a t e d compounds in Pediococcus c e r e v i s i a e . J . B a c t e r i o l . 104, 1-7.

8.

Lowry, O.H., N.J. Rosebrough, A.L. F a r r and R.J. R a n d a l l . 1951. P r o t e i n measurement with the Folin phenol reagent. J . B i o l . Chem. 193, 265-275.

9.

Mathews, C.K. and F.M. Huennekens. 1963. Further s t u d i e s on dihydrofolic r e d u c t a s e . J . B i o l . Chem. 238, 3436-3442.

and D.M. Moccio. Cancer Chemother.

BIOCHEMICAL AND CYTOTOXIC EFFECTS OF THE ERYTHRO- AND THREO-ISOMERS OF GAMMAFLUORO- METHOTREXATE

John J . McGuire

Grace Cancer Drug Center, Roswell Park Memorial Institute, Buffalo, NY

14263

John Galivan D i v i s i o n o f L a b o r a t o r i e s and Researcn, New Yorn ¿xaxe Department of Health, Albany, New York 12201 James K. Coward Department of Chemistry, Rensselaer Polytechnic Institute, Troy, NY

12181

Introduction

Gamma-fluoro-methotrexate (FMTX) i s a methotrexate (MTX) analog which contains 4-fluoroglutamate instead of glutamate. properties

as

MTX

itself

This analog has e s s e n t i a l l y the same

(dihydrofolate

reductase

inhibition,

uptake

and

e f f l u x , e t c . ) , except that FMTX forms polyglutamate derivatives poorly, i f a l l (1).

at

As such i t i s a useful tool to investigate the function of MTX poly-

glutamates.

This

previous

work

D,L-erythro:threo-4-fluoroglutamate D,L-erythro- and

was

performed

and i t

with

FMTX

synthesized

thus contained four

D,L-threo-4-fluoroglutamate

containing

from

isomers.

isomers

The

(eFMTX

and

tFMTX, respectively) have now been prepared and their properties determined.

Results

Pure eFMTX and tFMTX were separated from the mixed isomer population by HPLC. Separation was affected i s o c r a t i c a l l y (22°, 1.5 ml/min) on a Whatman P a r t i s i l 10 C-8 column (0.46 x 25 cm) equilibrated with 15 mM Na-phosphate, pH 2.2 containing

10% methanol.

The D,L-eFMTX

(tr

= 16.8 min)

and D,L-tFMTX

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

(tr =

862 19.6 min) were baseline resolved.

Each peak contained only one component when

re-analyzed under the same reverse phase conditions or by anion exchange HPLC. Pure

eFMTX

and

tFMTX

were

compared

to

MTX

as

inhibitors

of

dihydrofolate

reductase (DHFR) from a number of different sources.

For DHFR from H35 cells

[IC 5 Q ,nM=4.9

(MTX);

5.5

human

[IC S 0 ,nM=1.0

(MTX);

1.4

nM=l.1

(eFMTX); (eFMTX);

(MTX); 1.5 (eFMTX); 1.7

5.5 1.4

(tFMTX)], (tFMTX)],

and

leukemic

L1210

cells

(tFMTX)] the inhibition by either

essentially equivalent to that obtained with MTX

itself.

The

spleen [IC 5 Q ,

isomer was

slopes of the

inhibition curves were nearly identical for all drugs. The mixed isomer preparation of FMTX accumulated to the same level in cells as did MTX in short term (30 min) iiicuuations, Dut its initial rate of influx was slightly slower (1).

To assess the transport of the isomers of FMTX,

affect on the transport of 2 nM [ H]MTX was measured cell

line CCRF-CEM (Table I).

half

that

of of

the the

isomers

was

D,L-erythro

leukemia

Inhibition by the isomers was compared to the

isotopic dilution effect of unlabeled MTX. L-isomer

in the human

their

used

for

A concentration direct

and D,L-threo

pair

comparison should

be

of MTX since

that was only

inhibitory.

the The

presence of either 8 or 16 nM unlabeled MTX caused a decrease both in initial velocity and in the level D.L-eFMTX

well as did 8 transport. reduced

of accumulation at 30 min.

inhibited both the initial mM

MTX.

The tFMTX

velocity

The presence of 16 uM

and accumulation at 30 min as

isomer was much less inhibitory to [ 3 H]MTX

These results are consistent with eFMTX being transported by the

folate/MTX

system

with

kinetic

constants

itself while tFMTX probably has a lower affinity.

similar

to

those

The results also

of

MTX

indicate

that the reduced folate/MTX transporter is highly stereoselective. The eFMTX isomer is more cytotoxic than tFMTX against both H35

rat hepatoma

(ID 5 0

and

= 100

and 850 nM, respectively)

respectively) cell

and CCRF-CEM

(ID g 0

= 60

lines following continuous exposure, although both

170

nM,

isomers

are less potent than MTX itself (ID 5 Q = 9-15 nM). The effects of FMTX isomers on folate metabolism were assessed by looking at 3 the reduction of thymidylate synthesis from [ H]l)dR. Short exposure (4 hr) of CCRF-CEM cells to high concentrations 3 essentially

complete

inhibition

of

(2 >iM) of FMTX

[ H]UdR

isomers or MTX

incorporation.

fer of the cells to drug-free medium, incorporation

Following

gave

trans-

in the MTX treated cells

remained inhibited completely for 4 hr and did not even begin to recover until 8 hr.

This sustained inhibition is the result of polyglutamate synthesis dur-

863 Table I:

Effect of FMTX Isomers on [ H]MTX Transport by CCRF-CEM Cells Rate of I n i t i a l Addition

Accumulation at

Uptake pmol/min/10

30 Min

cells

pmol/10

None

0.153

2.55

8 tiM MTX

0.079

1.21

16 nM MTX

0.052

0.90

16 nM eFMTX

0.082

1.23

16

0.112

1.69

tFMTX

ing the i n i t i a l

exposure period (1).

In contrast,

cells

when FMTX treated c e l l s

were placed in drug-free medium, UdR incorporation began to recover by 1 hr and was essentially at control

rates by 4 hr.

Since the major

difference

between these two isomers and MTX i s that they do not form polyglutamates,

it

is most l i k e l y this property i s responsible for the difference in inhibitory behavior.

The isomers of FMTX showed the same kinetics

of

recovery

under

these conditions; however, they were clearly different when tested against H35 c e l l s at low drug concentrations at the long exposure times used in the cytotoxicity

assays.

The concentration

dependent

cytotoxicity

during

a 48 hr

exposure to each isomer wa^. compared to tne concentration dependence of hibition

of

3

[ H]H20

release

from

3

[5- H]UdR

during

the

first

24

hr

inof

exposure. There was a very close correspondence between cytotoxicity and 3 [ H]H,0 release profiles for each individual compound. However, comparing 3 the concentration required for 50% reduction in c e l l count and [ HjHgO release, the eFMTX isomer was about 5-fold more potent. Previous work with mixed isomer FMTX showed that only low levels of i t s polyglutamates could be detected.

The substrate activity of the individual FMTX

isomers with partially purified rat liver folylpolyglutamate synthetase (FPGS) was determined (Table I I ) .

Both isomers had activity near the detection limit

but i t appeared that eFMTX displayed s l i g h t l y higher activity than tFMTX. Higher FPGS substrate

activity

experiments

the

cells.

examining

of

eFMTX was also

metabolism

of

[14C]FMTX

indicated

in

preliminary

isomers

in

intact

H35

Following a 24 hr incubation in 1 ^M drug, there were 4.9 nmol poly-

864 Table II:

Substrate Activity of FMTX Isomers for Purified Rat Liver FPGS

4-NH 2 -10-CH 3 -Pte(F)Glu N Concentration

N =

2

3

4

pmol

Drug 8.5

MTX

17

341

39

2.7

646"

63

1.3

eFMTX

17

23.9

4.6

0

tFMTX

17

18.5

3.7

0

glutamates/mg

protein

from

eFMTX,

but

only

these same conditions MTX formed 23.7 nmol

2.1

nmol/mg

from

tFMTX.

Under

polyglutamates/mg.

The results on polyglutamate formation argue

that eFMTX

is a weak

for mammalian FPGS and that tFMTX is an even poorer substrate.

substrate

The increased

cytotoxicity and more sustained inhibition of jte novo thymidylate synthesis by eFMTX compared to tFMTX is apparently the result of its slightly higher level of

polygl utamate

polyglutamate classical

formation.

formation

is

These a

results

major

thus

determinant

reinforce of

the

the

notion

that

cytotoxicity

of

antifols.

Acknowledgements

This

work

was

supported

by Grants CA25933

Biomedical Research Support Grant.

and CA28097

from

the

NCI

and

a

JJM is a Leukemia Society Scholar.

References

1.

Galivan, J., Inglese, J., McGuire, J .J., Nimec, Z. and Coward, J.K. 1985. Gamma-fluoromethotrexate: Synthesis and Biological Activity of a Potent Inhibitor of Dihydrofolate Reductase With Greatly Diminished Ability to Form Poly-y-Glutamates. Proc. Natl. Acad. Sci. (USA) 82, 2598-2602.

C-^-TETRAHYDROFOLATE-S YNTHASE, A MULTIFUNCTIONAL ENZYME IN P U R I N E M E T A B O L I S M , A N D ITS RELATED M O N O F U N C T I O N A L

K.W. Shannon, T.R. Whitehead, C. Staben, J.C. D e p a r t m e n t o f B i o c h e m i s t r y , U n i v e r s i t y of Berkeley, California 94720

INVOLVED

ENZYMES

Rabinowitz

California,

Introduction The subject of this session thesis."

I would

activities

like

involved

is, " F o l a t e D e p e n d e n t P u r i n e

to d i s c u s s t h r e e p a r t i c u l a r

in t h e f o r m a t i o n of t h e o n e - c a r b o n

u s e d in p u r i n e b i o s y n t h e s i s . a historical manner

f i e l d of

to d e v e l o p

to d e s c r i b e h o w t h e e x p e r i m e n t a l

r e s u l t e d in u n a n t i c i p a t e d questions;

I w o u l d like

experimental

h a s a f f e c t e d t h e q u e s t i o n s w e ask a n d t h e a n s w e r s satisfactory.

This

as n o t o n l y r e l a t i n g previously

results,

b u t a l s o of s u g g e s t i n g

approach affected

w a s the influence of tracers.

Studies with compounds

L5

labeled with ' N showed that purine bases

are not derived from presynthesized 14 lty of

C made

find

However,

I

task some

questions experimen-

disposal.

A n e x a m p l e of h o w e x p e r i m e n t a l cally

the

interpreted my

i n t e r e s t to m e t h a t w e c o u l d a n s w e r w i t h

tal m e a n s a t our

to

approaches

t h e p r e s e n t to the p a s t a n d d e s c r i b i n g

unreported

of particular

so I h a v e

other

that we

is a " S t a t e o f the A r t L e c t u r e . "

am not sure just w h a t that means,

in

work

results that suggested still

and the p o s s i b l e

donors

the topic

h o w our e x p a n d i n g v i e w of a r e a s a p p r o p r i a t e

"biochemistry"

Syn-

enzymatic

it p o s s i b l e

sources

to d e m o n s t r a t e

investigation in r a t

(1).

The

isotopitissues availabil-

that uric acid,

the

purine e x c r e t e d by pigeons, w a s d e r i v e d from small m o l e c u l e s as carbon dioxide and glycine

(2-4).

The

fact that formate

s h o w n to b e a n e x t r e m e l y e f f e c t i v e p r e c u r s o r

of p u r i n e

carbon

a t o m s 2 a n d 8 in u r i c a c i d

(2) w a s a n u n e x p e c t e d

formate had not previously

been recognized as a metabolic

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

such was

finding

since intermediate.

866 E f f o r t s w e r e then d i r e c t e d t o w a r d d e f i n i n g the i n d i v i d u a l

meta-

b o l i c r e a c t i o n s i n v o l v e d in the s y n t h e s i s of h y p o x a n t h i n e

and

i n v o l v e d the use of p i g e o n liver h o m o g e n a t e s

(5).

Through

the 14

use of this s y s t e m for m e a s u r e m e n t s of the i n c o r p o r a t i o n of

C-

f o r m a t e , it w a s r e c o g n i z e d that I n o s i n i c acid w a s the

first

p u r i n e f o r m e d in the iji v i t r o s y s t e m

responsible

(6).

The enzyme

for the " a c t i v a t i o n " of f o r m a t e w a s s h o w n to r e q u i r e A T P tetrahydrofolic acid 10-formyl-THF formylase" nized.

(8,9).

(9).

(THF)

(7) a n d to r e s u l t in the f o r m a t i o n of

It w a s o r i g i n a l l y n a m e d ,

"anhydroleucovorin"

(5-formyl-THF)

or 5 , 1 0 - m e t h e n y l - T H F

recog-

plus ATP

(7), a n d

(13,14)

I w i l l l i m i t my d i s c u s s i o n to the e n z y m a t i c

(10),

serine

S u b s e q u e n t work e s t a b l i s h e d the e n z y m a t i c

i n v o l v e d in the u t i l i z a t i o n of 5 - f o r m y l - T H F (15).

"tetrahydrofolate

O t h e r s o u r c e s of the a c t i v e f o r m a t e w e r e

These included leucovorin

(5,11,12).

and

reactions

and

serine

reactions

r e l a t e d to the a c t i v a t i o n of formate by t e t r a h y d r o f o l a t e

for-

m y l a s e , w h i c h w e n o w c a l l 1 0 - f o r m y l - T H F s y n t h e t a s e and the c o n v e r s i o n of 1 0 - f o r m y l - T H F to o t h e r d e r i v a t i v e s u s e d in p u r i n e biosynthesis.

The

Prokaryotes

T h e e x p e r i m e n t a l s y s t e m s I h a v e d e s c r i b e d for i n v e s t i g a t i n g m e c h a n i s m s of p u r i n e b i o s y n t h e s i s w e r e a u g m e n t e d by work use of other c o n c e p t s t h a t w e r e less o b v i o u s l y a p p l i c a b l e answer this p r o b l e m .

to

T h e y i n c l u d e d the use of b a c t e r i a and

d i e s o n the d e g r a d a t i o n rather than the b i o s y n t h e s i s of T h e i n v e s t i g a t i o n of p u r i n e f e r m e n t a t i o n by a n a e r o b i c

the

making stu-

purines.

organisms

(16) w a s d e p e n d e n t o n the i n f o r m a t i o n a n d t e c h n i q u e s d e r i v e d

from

the work o n p u r i n e s y n t h e s i s in p i g e o n s for the s y n t h e s i s of 14 C-uric acid

(17).

T h e l a b e l l e d u r i c acid w a s used to d e t e r m i n e

the o r i g i n of the p r o d u c t s of this f e r m e n t a t i o n a c i d w a s i d e n t i f i e d as o n e of these p r o d u c t s

(18).

Formic

(19) and the

reac-

t i o n s i n v o l v e d in its f o r m a t i o n w e r e d e t e r m i n e d t h r o u g h the use of c e l l - f r e e s y s t e m s

(20) .

The enzyme 10-formyl-THF

synthetase,

867 originally named tetrahydrofolate formylase, responsible for the activation of formate: Formate

+ THF + MgATP

-2

->

10-formyl-THF + MgADP

+ HPO

4

-2

was purified from Peptococcus asaccharolyticus (ATCC 14963) (previously called Micrococcus aerogenes and Peptococcus aerogenes) (21,22), and from four clostridial species (23-26).

It was

obtained in crystalline form from Clostridium cylindrosporum (23).

These bacterial enzymes are homotetramers of M r = 60,000.

The physical and kinetic properties of the enzymes and the mechanism of the reaction have been reviewed (27,28) and will not be discussed here. It should be noted that these bacterial enzymes are derived from organisms in which the enzyme occurs in exceptionally high levels and is believed to catalyze reactions other than those involved in the synthesis of purine bases.

These are for the generation

of ATP from 10-formyl-THF in the fermentation of a purine substrate, or in the utilization of CO. after its reduction.

Yeast and Other Eukaryotes Partially purified 10-formyl-THF synthetase from pigeon liver (29) and sheep liver (30), were used in studies on the mechanism of the reaction.

The observation that the 10-formyl-THF synthe-

tase activity of porcine liver copurified with two other enzymes, methenyl-THF cyclohydrolase and methylene-THF dehydrogenase:

HCOOH

H*N N (I r T I

ir imirDP H2N

—

n

N

XXX ¡0 - For my! — tetrahvdrofulaie

Synthetase

NADPH NADP® HjN

pQ*

OH

CHO (B)

»"(A) TelruhyJrofulate

HiN M

un

5.10 • Meihenyl — tetrah ydrofotate

Cyclohydrolase

CHj '

5.10 - Methylene lelrahydro/o/oie

Dehydrogenase

-

868 led to the s u g g e s t i o n that these e n z y m e s o c c u r r e d as a c o m p l e x (31).

G e n e t i c e v i d e n c e s u g g e s t e d that the y e a s t A P E 3

gene

e n c o d e d a t r i f u n c t i o n a l p r o t e i n w i t h the 3 a c t i v i t i e s s h o w n above (32,33).

T h e s e o b s e r v a t i o n s s t i m u l a t e d the r e c o g n i t i o n that the

h o m o g e n e o u s 1 0 - f o r m y l - T H F s y n t h e t a s e s a l r e a d y i s o l a t e d from o v i n e (34) a n d p o r c i n e

(35) liver w e r e t r i f u n c t i o n a l p r o t e i n s w i t h

the

three a c t i v i t i e s s h o w n a b o v e and s t i m u l a t e d the p u r i f i c a t i o n of the e n z y m e from S a c c h a r o m y c e s c e r e v i s i a e

(36).

The

trifunctional

p r o t e i n s are d i m e r i c h o m o p o l y m e r s w i t h s u b u n i t s of M r = 100 118

(28).

This molecular

structure

is d i f f e r e n t from that w h i c h

o c c u r s in the p u r i n o l y t i c b a c t e r i a w h e r e the three o c c u r as s e p a r a t e m o n o f u n c t i o n a l e n z y m e s . forms occur

that w i l l be d i s c u s s e d shortly

molecular

(28). polyfunctional

T h e r e f o r e , w e use the name

synthase" for this t r i f u n c t i o n a l e n z y m e . methylene-THF synthase(combined)

"C^-THF

W e find that o t h e r

n a m e s that h a v e b e e n u s e d in the p a s t , such as use.

activities

B u t other

U n f o r t u n a t e l y , n o s t a n d a r d i z e d n o m e n c l a t u r e for e n z y m e s has b e e n e s t a b l i s h e d .

to

formyl-methenyl-

are too c u m b e r s o m e for

general

W e feel that some g e n e r a l c o n v e n t i o n is r e q u i r e d to d i f -

ferentiate multifunctional functional

e n z y m e s from their c o r r e s p o n d i n g

mono-

forms.

C ^ - T H F s y n t h a s e is not the o n l y m u l t i f u n c t i o n a l e n z y m e

involved

in p u r i n e b i o s y n t h e s i s .

proteins

A t l e a s t four m u l t i f u n c t i o n a l

are a s s o c i a t e d w i t h p u r i n e b i o s y n t h e s i s de novo: tional C^-THF synthase, a bifunctional

(37) and

(38) e n z y m e e a c h w i t h one of the r i b o n u c l e o t i d e a c t i v i t i e s and an a d d i t i o n a l b i f u n c t i o n a l e n z y m e ingly, one of the m u l t i f u n c t i o n a l that are n o t all s e q u e n t i a l

Yeast

the

trifunc-

trifunctional transformylase (39).

enzymes catalyzes

Interest-

reactions

(38).

Mitochondria

In s t u d i e s d e m o n s t r a t i n g

t h a t A P E 3 is the s t r u c t u r a l g e n e for S.

cerevisiae C^-THF synthase

(40), w e n o t e d that an ade3

s t r a i n h a d low b u t d e t e c t a b l e

levels of 1 0 - f o r m y l - T H F

deletion synthetase

869 and 5,10-methylene-THF dehydrogenase

(41).

In order

the nature of these a c t i v i t i e s , we p u r i f i e d the

f r o m t h e d e l e t i o n m u t a n t as s h o w n in T a b l e

activity

to

determine

synthetase I.

T a b l e I. PURIFICATION OF f-THF SYNTHETASE FROM AN ade3 DELETION STRAIN

f - T H F synthetase activity

Step

Crude extract Protamine Sulfate Ammonium Sulfate C M cellulose Heparin Agarose Blue Sepharose Hydroxylapatite

Protein mg

Total IU

S.A. lU/mg

Purif. fold

Yield

%

SYN DH

SYN CYC

40,000 30,000 9,400 180 20 3.1 1.6

600 600 570 300 220 100 94

0.015 0.020 0.060 1.7 11 33 59

1.0 1.3 4.0 110 720 2200 3900

100 100 95 51 36 17 16

4 3 3 5 9 8 4

45 14 16 11 13 14 15

We obtained a protein that we estimate in a 16% y i e l d a f t e r cyclohydrolase

to b e m o r e t h a n 90%

4000-fold purification.

encoded by A P E 3 ,

that this protein,

i s o z y m e h a s a M^ = 1 0 0 , 0 0 0 w h i c h

SDS-PAGE

is s i m i l a r

THF synthase.

Gel exclusion chromatography

tein has an M r

= 200,000

identical

suggesting

s u b u n i t s a s is C ^ - T H F

T h e p r e s e n c e o f the e n z y m e the structural g e n e isolated an isozyme. enzyme differed

to t h e v a l u e

for

shows that the

t h a t it is c o m p o s e d of

suggested that we

to c o n f i r m t h e f a c t t h a t

T h e p e p t i d e b a n d i n g p a t t e r n s of

t h e m u t a n t e n z y m e as a n i s o z y m e of C ^ - T H F

We also used immunological r e l a t e d n e s s of t h e s e

techniques

isozymes.

We

purified enzymes with polyclonal of the p u r i f i e d e n z y m e s

protwo

in a y e a s t m u t a n t w i t h a d e l e t i o n

for C ^ - T H F s y n t h a s e In order

(Fig.

the C^-

synthase.

e n z y m e s a r e d i f f e r e n t a n d t h e s e r e s u l t s s u p p o r t our of

synthase

shows that

1).

of

had

this

from C ^ - T H F synthase, we c o m p a r e d the two

by peptide mapping.

and

synthetase

like the C ^ - T H F

is a l s o t r i f u n c t i o n a l .

pure

Dehydrogenase

a c t i v i t i e s c o p u r i f i e d w i t h the

activity, suggesting

S.A. ratios

the

enzymes two

designation

synthase.

to d e t e r m i n e

the

structural

immunotitrated each of

antlsera directed against

the each

870 100

80 60 |

s

40

%

20

•>

100

o

o


H

X

S

R e s t r i c t i o n m a p of p T W 2 .

T h e pUC8 D N A is s h o w n by the thick l i n e , a n d the g e n o m i c by the thin line.

T h e s q u a r e i n d i c a t e s the site of

of s y n t h e t i c o l i g o n u c l e o t i d e s p r e p a r e d from c o d o n s for a c i d s 10-15 of the p r o t e i n .

insert

hybridization amino

T h e a r r o w i n d i c a t e s the l o c a t i o n of

the s y n t h e t a s e g e n e a n d the d i r e c t i o n of t r a n s c r i p t i o n of synthetase message.

d i a l e n z y m e g e n e , a n d h a v e use this s e q u e n c e to d e t e r m i n e corresponding

the

W e h a v e s e q u e n c e d a b o u t 75% of the c l o s t r i -

a m i n o acid s e q u e n c e

the

(Fig. 6).

• DH/CYC— 1

320

469 523 586 S. cerevisiae C -THF Synthase

I

nh

150

151

214

1 h+

3

C. acid-urici 10-Formyl-THF Synthetase COOH

Direct A m i n o Acid Homology

53%

53%

38%

+ AA Substitution

59%

62%

44%

Fig. 6.

A m i n o acid h o m o l o g y b e t w e e n S. c e r e v i s i a e C ^ - T H F syn-

t h a s e and and C. a c i d i - u r i c i

10-formyl-THF

synthetase.

877 T h e bold p o r t i o n of the lower line i n d i c a t e s the p o r t i o n of c l o s t r i d i a l p r o t e i n t h a t we h a v e s e q u e n c e d .

The dashed

the

line

s h o w s the p o r t i o n of the s e q u e n c e that r e m a i n s to be d e t e r m i n e d . T h e d e g r e e of h o m o l o g y to the p o r t i o n s of the y e a s t C ^ - T H F t h a s e i n d i c a t e d are g i v e n b e l o w those p o r t i o n s .

syn-

The homology

is

near 60% for the two s e g m e n t s near the N - t e r m i n a l p o r t i o n , if o n e o m i t s a s h o r t p o r t i o n of the y e a s t e n z y m e .

The carboxy

s h o w s s o m e w h a t less h o m o l o g y , b u t e v e n that is 44%.

portion

We have

n o t e d t h a t the p o r t i o n of the y e a s t e n z y m e b e t w e e n these

two

h o m o l o g o u s p o r t i o n s is q u i t e h i g h in b a s i c a m i n o acids.

This

d e g r e e of h o m o l o g y is u n u s u a l for p r o t e i n s from s o u r c e s as d i v i r g e n t as a b a c t e r i u m a n d y e a s t . entire sequence very

I b e l i e v e t h a t we w i l l have

the

shortly.

W e h a v e c l o n e d y e a s t D N A that h y b r i d i z e s to d e g e n e r a t e

oligonu-

c l e o t i d e p r o b e s d e r i v e d from the amino t e r m i n a l s e q u e n c e of yeast mt-C^-THF synthase.

the

I n a d d i t i o n to s e q u e n c i n g the m t - C ^ -

T H F s y n t h a s e g e n e in o r d e r to d e t e r m i n e the a m i n o a c i d

sequence

of the e n z y m e a n d its r e l a t i o n s h i p to the c y t o p l a s m i c p r o t e i n , h o p e to c r e a t e a m u t a n t in this g e n e by g e n e d i s r u p t i o n .

m u t a n t c o u l d g i v e i n s i g h t to the n o r m a l f u n c t i o n of the g e n e . a d d i t i o n , o v e r e x p r e s s i o n of the p r o t e i n w o u l d m a k e it easier isolate the p r o t e i n a n d c h a r a c t e r i z e

the g e n e a n d the

we

Such a In to

protein.

Occurrence and Significance N o w that w e r e c o g n i z e that 1 0 - f o r m y l - T H F s y n t h e t a s e a c t i v i t y occur

in d i f f e r e n t m o l e c u l a r

forms, and e v e n d i f f e r e n t

can

isozymic

f o r m s w i t h i n o n e o r g a n i s m , I b e l i e v e that it is of i n t e r e s t

to

c o n s i d e r w h a t is k n o w n a b o u t the o c c u r r e n c e of these forms among various organisms.

T h e e n z y m e w a s f i r s t d e s c r i b e d in 1955 b a s e d

o n its a c t i v i t y in pig a n d p i g e o n liver, a n d w a s a s s u m e d to be monofunctional

(8,9), as w a s the c r y s t a l l i n e e n z y m e d e r i v e d

b a c t e r i a in 1958 (27).

(49) .

T h e a c t i v i t y has w i d e s p r e a d

T h e s u g g e s t i o n t h a t the a c t i v i t y o c c u r s in a s e c o n d

associated

(in an u n s p e c i f i e d manner)

from

occurrence form

with other enzymes was

f i r s t m a d e in 1972 a n d 1973 b a s e d o n g e n e t i c a n d

biochemical

878 evidence (31,32).

The occurrence of the monofunctional

form,

10-formyl-THF synthetase, has been established in a number of bacteria (28) .

The trifunctional form of the enzyme, C^-THF syn-

thase, is found in pig, sheep, cow, chicken and rabbit liver and in yeast (28).

It is thought to function primarily in purine

nucleotide synthesis.

Is it possible that the purine utilizing

bacteria contain a trifunctional protein in addition to the monofunctional 10-formyl-THF synthetase?

It should be noted that the

monofunctional enzyme has been isolated only from sources that contain the enzyme in concentrations about 2500 times higher than are found in most other bacteria that we have examined.

This may

result from the enzyme's involvement in reactions related to the utilization of the carbon source provided rather than exclusively for biosynthetic reactions.

Is it possible that we have over-

looked the presence of a trifunctional enzyme that might occur in concentrations that are exceedingly low relative to the amount of monofunctional enzyme in these particular cells?

This does not

appear to me to be such a far-fetched possibility in view of the very highly organized and complicated nature of the enzymatic components required for purine nucleotide biosynthesis in animal systems where it seems plausible that the structure of each element involved is essential for the activity

(37).

When we consider how representative these examples are of the organisms representing life on this earth, I think we must recognize how inadequate our present state of knowledge is since these examples do not include any examples from Protoctista or Plantae and are obviously inadequate even with respect to the kingdoms that have been e x a m i n e d — t h e Monera, Animalia and Fungi

(50).

Without pretending to tackle this problem, but responding to the stimulation in considering it, we have decided to determine the nature of this enzyme in various species with these considerations in mind.

Our approach to the problem has been to consider

the phylogenetic classification of life, to try to choose examples that represent some variety within each kingdom and that are available to us, and to first answer the simple question of whether the 10-formyl-THF synthetase activity is associated with

879 a monofunctional enzyme or with one possessing either or both the dehydrogenase and/or cyclohydrolase.

The experimental means for

doing this is based on our observation that heparin-agarose appears to bind enzymes that use THF or its derivatives as a substrate.

Thus, by passing a crude extract of the cell over such a

column and eluting it with a salt gradient, we can separate the three activities when they occur as monofunctional enzymes.

If

we detect all three activities in a single peak, this suggests that the enzyme occurs in a polyfunctional form. an extract of C. acidi-urici

The behavior of

(Fig. 7) shows that the enzymes in

this source are monofunctional proteins.

Analysis of an extract

of yeast shows a single peak with these three enzymic activities eluting with about 150 mM KC1.

Fraction Number (5 ml) Fig. 7.

Chromatography of extract of C. acidi-urici on heparin-

agarose. If we consider just the kingdom Monera (bacteria), we realize that our knowledge concerning the organization of is based on the analysis of very few species.

We have started to examine a group

of bacterial species that we believe represents a reasonable cross-section of organisms (Table III).

880 Table

III.

SURVEY OF PROCAYOT1C ORGANISMS FOR IO-FORMYL-THF SYNTHETASE GRAM-POSITIVE ORGANISMS: A. Aerobic Rods: Bacillus subtilis B. Aerobic Cocci: Micrococcus luteus C. Facultative Cocci: Staphylococcus epidermidis Streptococcus faecalis DL Anaerobic Rods: Clostridium absonum Clostridium pasteurianum Eubacterium sp. V.P.I. 12708 E Anaerobic Cocci: Peptostreptococcus anaerobius F. Actinomycetes: Streptomyces lividens

II. GRAM-NEGATIVE ORGANISMS: A. Aerobic Rods: Pseudomonas aeruginosa B. Aerobic Cocci: Branhamella catarrhalis C. Facultative Rods: Escherichia coli Salmonella typhimurium Klebsiella pneumonia Proteus mirabilis Shigella tlexneri D. Anaerobic Rods: Bacteroides fragile III. ARCHAEBACTERIA: A. Aerobic Rods: Halobacterium salinarium

T h i s s t u d y is j u s t s t a r t i n g b u t I w o u l d like to m e n t i o n some of the r e s u l t s t h a t we h a v e o b t a i n e d in our e x a m i n a t i o n of e n t e r i c b a c t e r i a s h o w n in T a b l e Table

the

IV.

IV. SPECIFIC ACTIVITY OF IO-FORMYL-THF SYNTHETASE AND 5,10METHYLENE-THF DEHYDROGENASE IN CRUDE EXTRACTS OF VARIOUS BACTERIA Organism I. Gram-Negative A. Aerobic Rods: Pseudomonas aeruginosa Facultative Rods: Escherichia coli Klebsiella pneumonia Proteus mirabilis Shigella flexneri Salmonella typhimurium Anaerobic Rods: Bacteroides fragiiis II. Gram-Positive A. Anaerobic Rods Clostridium acidi-urici Clostridium pasteurianum

Synthetase (mU/mg)

Dehydrogenase (mU/mg)

0

10

0 0 12 0 0

25 19 30 58 32

100

19

13,000 60

600 89

W e c a n n o t d e t e c t the s y n t h e t a s e

in E. c o l i in a g r e e m e n t w i t h p r e -

v i o u s r e p o r t s for this o r g a n i s m

(43,51).

Nor d o we d e t e c t

the

s y n t h e t a s e in any of the other b a c t e r i a of this g r o u p , e x c e p t for Proteus mirabilis.

A l l of these o r g a n i s m s d o c o n t a i n

the

881 dehydrogenase.

It remains to be determined whether it is mono-

functional or is associated with cyclohydrolase in a bifunctional enzyme as reported for E. coli

(51).

Also in this table is the

value for synthetase found in Clostridium pasteurianum.

This

organism was of interest to us because other clostridial

species

that have been examined all contain exceptionally high levels of this monofunctional enzyme, and all have unusual one-carbon metabolism.

C. pasteurianum is a saccharolytic organism that is not

unusual with respect to the metabolism of formate.

It has a much

lower level of the synthetase as well as the dehydrogenase does the purinolytic Clostridium.

than

We considered the possibility

that the activity might be associated with a trifunctional protein involved in purine biosynthesis.

In fact, is this the basis

for the distinction between the monofunctional and trifunctional forms of the enzyme?

As shown in Fig. 9, the enzymes appear to

be monofunctional in this organism.

Fraction Number (2 ml) Fig. 9.

Chromatography of extract of C. pasteurianum on

heparin-agarose. We would also like to examine representatives of other for the state of these enzymes.

kingdoms

Synthetase activity has been

reported i n plants (52) , but the functionality of the enzyme is not known.

A preliminary examination of a crude homogenate of

whole spinach leaves suggests that the enzyme is trifunctional

882 (Pig. 13, J. Nour, unpublished observations).

200

-o- Synthetase


• cob( II )alamin + N2 + OH.

[3]

The hydroxyl radical could attack amino acid residues near the active site of the enzyme, or the radical could attack the bridge carbon of the corrin, which would interrupt conjugation and form a xanthocorrinoid

(12); either reaction would lead to an irrevers-

ible inactivation of methionine synthase.

We also observed

the

loss of protein after turnover under N2O; this could also be the result of the cleavage of the protein by hydroxyl radical generated at the active site

(13).

Acknowledgement The research was funded by National Institutes of Health Grant GM24908.

References 1.

Dolphin, D.

1971.

Methods Enzymol. 18C, 34-52.

2.

Taylor, R.T., H. Weissbach. 1502-1508.

1967.

J. Biol. Chem. 242,

3.

Fujii, K., F.M. Huennekens. 6745-6753.

1974.

J. Biol. Chem. 249,

4.

Bayston, J.H., F.D. Looney, J.R. Pilbrow, M.E. Winfield. 1970. Biochemistry 9, 2162-2172.

5.

Firth, R.S., H.A.O. Hill, B.E. Mann, J.M. Pratt, R.J.P. Williams. 1967. J. Chem. Soc. Chem. Commun. 1013.

6.

Hayward, G.C., H.A.O. Hill, J.M. Pratt, R.J.P. Williams. 1965. J. Chem. Soc. 6485.

920 7.

Fujii, K. , F.M. Huerinekens. 1979. In: Biochemical Aspects of Nutrition (K. Yagi, ed.) Japan Scientific Societies Press, Tokyo, pp. 173-184.

8.

Beinert, H., G. Palmer. 1965. In: Oxidases and Related Redox Systems, Vol. 2 (T.E. King, H.S. Mason, and W. Morrison, eds.) Wiley, New York, pp. 567-590.

9.

Deacon, R., M. Lumb, J. Perry, I. Chanarin, B. Minty, M.J. Halsey, J.F. Nunn. 1980. Eur. J. Biochem. 104, 419-422.

10. Banks, R.G.S., R.J. Henderson, J.M. Pratt. Soc. (A), 2886-2889. 11. Blackburn, R., M. Kway, A.J. Swallow. Farad. Trans. 21, 250-255. 12. Bonnett, R. 1982. In: New York, pp. 202-243.

1968.

1977.

J. Chem.

J. Chem. Soc.

Vol. 1 (D. Dolphin, ed.) Wiley,

13. Kim, K., S.G. Rhee, E.R. Stadtman. 15394-15397.

1985.

J. Biol. Chem. 260,

Inhibition of Methylenetetrahydrofolate Reductase by Adenosylmethionine

David A. Jencks and Rowena G. Matthews Biophysics Research Division and Department of Biological Chemistry University of Michigan, Ann Arbor, Michigan 48109

Introduction We have studied the allosteric regulation of the dimeric flavoprotein methylenetetrahydrofolate reductase from pig liver by the inhibitor adenosylmethionine (AdoMet) and the substrate NADPH.

Methylenetetrahydrofolate reductase catalyzes the NADPH-linked reduction of

methylenetetrahydrofolate [eq.l].

Methyltetrahydrofolate serves as a methyl donor for the > NADP* + CH 3 -H 4 folate

NADPH + CH 2 -H 4 folate methylation

of

homocysteine in the reaction

catalyzed

[1]

by 5-methyltetrahydrofolate-

homocysteine methyl transferase, and the combined action of methylenetetrahydrofolate reductase and 5-methyltetrahydrofolate-homocysteine methyl transferase supplies one carbon units for methylation reactions using AdoMet. The reaction [1] proceeds by a ping-pong bi-bi mechanism, and we have taken advantage of this to study the NADPH half-reaction by measuring electron transfer from NADPH to menadione. Electron transfer from the enzyme to menadione is second order, so the NADPH half reaction is rate limiting at appropriate menadione concentrations1.

The inhibition caused by AdoMet takes several minutes to reach its full extent, and so we have been able to study not only the activity at equilibrium of enzyme, AdoMet and NADPH, but also to study the kinetics of approach to equilibrium. As a result of this, we are able to propose a model for the interactions of methylenetetrahydrofolate reductase, AdoMet and

NADPH

involving not only the equilibrium constants between various forms of the enzyme but also some of the microscopic rate constants.

Results We have conducted our experiments by measuring the activity of methylenetetrahydrofolate reductase after incubation of the reduced enzyme with AdoMet and NADPH. We used the

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

922 NADPH-menadione assay to measure activity. In this assay the enzyme is primarily oxidized during steady state turnover1. For equilibrium experiments, a 20 minute preincubation was used, whereas for kinetic experiments, the activity was measured at time points up to 20 minutes.

In one set of experiments the NADPH concentration during the assay was 200 |iM. We find that at concentrations of NADPH in the preincubation up to 200 |iM the dependence of activity on [AdoMet] appears hyperbolic, but that at NADPH concentrations of 1 to 2 mM sigmoidicity appears, indicating the presence of cooperative interactions in AdoMet binding. Furthermore, the activity in the absence of AdoMet rises with increasing NADPH concentration in the preincubation, suggesting that there are two states of the enzyme with differing affinities for NADPH, the form with higher affinity also having higher activity.

When the assay is

conducted at the same NADPH concentration as the preincubation, we again observe that increasing [NADPH] overcomes the inhibition caused by AdoMet and high levels of NADPH appear to increase the amount of active enzyme present.

The kinetics of inhibition by AdoMet are pronouncedly biphasic, with the predominant effect of increasing levels of AdoMet being to increase the extent of the fast phase, although some increase in the rate of the slow phase is also seen. NADPH again has an antagonistic effect, whereby, at high AdoMet concentrations, increasing the NADPH concentration decreases the extent of the fast phase and decidedly reduces the rate of the slow phase.

Since the cooperativity of AdoMet binding suggests that there is more than one AdoMet binding site, we conducted a spectrophotometric titration of methylenetetrahydrofolate reductase, using the flavin as a probe, and found that there is indeed one AdoMet binding site for each subunit.

Model for methylenetetrahydrofolate reductase The model we propose for the interactions of methylenetetrahydrofolate reductase with AdoMet and NADPH is similar to the model for allosterism originally proposed by Monod, Wyman, and Changeux 2 . Thus there are two quaternary states, R and T, to which the ligands bind with different affinities; NADPH more tightly to the R state, AdoMet more tightly to the T state. As in an MWC model, this produces homotropic and heterotropic cooperative effects. For instance, as [AdoMet] increases, AdoMet ligated R and T state enzyme become more stable, but due to the greater affinity for T state enzyme, AdoMet ligated T state enzyme

923 eventually becomes the most stable form and accumulates. Since T state enzyme has little activity, this is manifested as inhibition. An MWC model of this kind adequately explains the equilibrium data. However, an MWC model predicts that the kinetics of inhibition will be a single first order process if ligand binding is rapid equilibrium, and will exhibit a lag phase if ligand binding is on a finite time scale. Thus the rapid burst of inhibition we observe at high levels of AdoMet is inconsistent with an MWC model. In order to explain the biphasic kinetics of inhibition, we propose that, on a given subunit in a given quaternary state, binding of one ligand considerably reduces the affinity for the other. AdoMet binding to the R state occurs at a rate commensurate with the burst phase of inhibition, and R—>T conversion at a rate commensurate with the slow phase. The biphasic kinetics is then a result of relatively rapid binding of AdoMet to R state enzyme, thus excluding NADPH binding and hence activity, followed by slow conversion to the thermodynamically more stable AdoMet ligated T state. At high NADPH concentrations, NADPH binds to R state enzyme, thus reducing its availability for AdoMet binding and decreasing the extent of the fast phase; NADPH ligated R state forms do not readily convert to T state, so the reduction of the amount of free R state enzyme also reduces the rate of the slow-phase R—>T conversion.

An additional complication is presented by the differing NADPH affinities of methylenetetrahydrofolate reductase in reduced and oxidized forms, here present during the preincubation and the assay respectively. We are able to take account of this and construct a mathematical model embodying the above features that adequately explains all our equilibrium and kinetic data.

Conclusion We find that AdoMet and NADPH interact antagonistically with methylenetetrahydrofolate reductase with respect to both the equilibria between active and inactive enzyme forms and the kinetics of approach to that equilibrium. AdoMet inhibits the enzyme and increases the extent of the fast phase of the biphasic inhibition, whereas NADPH tends to counteract this inhibition and reduces the extent of the fast phase as well as the rate of the slow phase.

We have

constructed a mathematical model that quantitatively predicts our equilibrium and kinetic data.

References

1. Vanoni, M. A., Ballou, D. P., and Matthews, R. G. (1983) J. Biol. Chem. 258, 11510-11514 2. Monod, J., Wyman, J., and Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118

REGULATION OP FOLATE HOMEOSTASIS

C. Osborne, K. Lowe, B. Shane Department of Nutritional Sciences, University of California, Berkeley, California 94720 D. J. Cichowicz, D. Sussman, G. Milman Department of Biochemistry, The Johns Hopkins University, Baltimore, Maryland 21205

Introduction Folate retention by tissues is dependent on conversion to p o l y glutamate derivatives, catalyzed by folylpolyglutamate synthetase (FPGS).

Factors i n v o l v e d in the regulation of folate homeostasis

are under study with an in vitro model, using homogeneous pig liver FPGS (1), and an in x i ¥ 2 model, using cultured mammalian cells, including Chinese hamster ovary c e l l FPGS mutants expressing the human FPGS gene.

Some preliminary results are presented.

Results Table 1 shows kinetic constants of H 4 P t e G l u n for pig liver FPGS. Increasing the glutamate chain length of H^PteGlu causes a decrease in k c a t but KJJ, v a l u e s remain low.

The retention of

affinity by polyglutamates, which drops off beyond the pentaglutamate, suggests that long chain length folates that accumulate in c e l l s h a v e the potential of regulating the synthesis of f o l y l p o l y glutamates.

Polyglutamates of other reduced folates also retain

high affinity for the enzyme but their k c a t values drop off more rapidly and only H 4 P t e G l u polyglutamates can be converted to the long chain length derivatives that accumulate in tissues. Competition between competing substrates is shown in Table 2.

The

pig liver enzyme metabolizes low concentrations of H 4 P t e G l u to the same types of derivatives that are found i n 3£iY2> with the hexa-

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

926 Table 1 Substrate

Kinetic Constants of Folylpolyglutamate Synthetase (nM)

THF THF(Glu-2) THF(Glu-3)

7.7 3.4 1.1

THF(Glu-4) THF(Glu-5) THF(Glu-6)

2.0 2.7

THF(Glu-7)

K^ (|iM)

11 14 34 47

V m a x (rel)

V m a x /K m (r

100 45 8.8

100 102 62

4.5 1.6

17 5 1000

Km ( U M ) ± SE

± SE (nM)

k

(min - 1 ) ± SE

Acknowledgement This

research was

CA18856

supported

(FMS) , and CA22764

by PHS grant numbers CA25236 (FMS)

awarded

by

the National

(JRP), Cancer

institute, DHHS.

References 1.

Piper, J. R., McCaleb, G. S., Montgomery, J. A., Kisliuk, R. L., Gaumont, Y., Sirotnak, F. M. 1986. J. Med. Chem. 21, 1080.

2.

Schmidt, H. W., Junek, H.

3.

Elslager, E. F., Davoll, J. 2, 97.

4.

Temple, C., Jr., Elliott, R. D., Montgomery, J. A. Org. Chem. 47, 761.

5.

Taylor, E. C., Harrington, P. J., Fletcher, S. R., Beardsley, G. P., Moran, R. G. 1985, J. Med. Chem. 28, 914.

6.

Beardsley, G. P., Taylor, E. C., Shih, C., Poore, G. A., Grindley, G. B., Moran, R. G. 1986. Proc. Am. Assoc. Cancer Res., 1027.

1977.

Monatsch. Chem. 108, 895.

1974.

Lect.

Heterocycl. Chem. 1982.

J.

AZIDO-SUBSTITUTED ANTIFOLATE DRUGS: ACTIVITY

SYNTHESIS, STRUCTURE, AND

P.K. Bryant, K.P. Wong, J. Colby, C.H. Schwalbe, M.F.G. Stevens, R.J. Griffin, and E.A. Bliss Pharmaceutical Sciences Institute, Department of Pharmaceutical Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, England

Introduction Lipophilic inhibitors of dihydrofolate reductase (DHFR) can enter lipid compartments such as the brain, and cancer cells able to exclude more polar antifolates such as methotrexate. However, 2 many have long biological half-lives, e.g. a plasma tjy2 h (1) for 2,4-diamino-5-(3,4-dichlorophenyl)-6-methylpyrimidine (metoprine, DDMP), resulting in slow clearance of any toxic effects which develop. The aromatic azido substituent, as in compounds offers an attractive alternative: it is lipophilic but it can be transformed to a more polar amino group, aiding elimination.

1

2

3

Results and Discussion Synthesis of _l-_3 from the appropriate nitro compounds involved reduction with hydrazine-Raney nickel or stannous chloride, followed by diazotisation-azidation of the resulting amine in acid solution by adding first NaNO_ and then NaN- solutions.

Chemistry and Biology of Pteridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

1006

The azido group in l-v3 can react in several ways. The sulphydryl reagents solium hydrosulphide, 2-mercaptoethanol, and dithiothreitol all reduced 1 smoothly to the arylamine. Thus it is likely that cellular thiols can effect a similar reduction in vivo. Heating 1 and 2^ with hydrazine hydrate completely removed the azido group leaving only hydrogen at that position, a reaction which may be of synthetic utility. Similar treatment of 3 yielded 2,4-dihydrazinoquinazoline. Treatment of _1 with trifluoromethanesulphonic acid at 0° caused rapid evolution of nitrogen and introduction of a bulky CF^SO^ substituent into the hindered 2'-position of the phenyl ring instead of intramolecular cyclisation. Thermolysis of or photolysis in nitrogensaturated water converted the azide to an amine, while photolysis in oxygen-saturated water produced the nitro compound. Thermolysis in nitrobenzene or dekalin and photolysis in water or methanol of the free base of 2 yielded only products derived from the triplet nitrene intermediate: 2,4,6-triaminoquinazoline and a maroon azo-dye. No singlet-derived ring-expanded products were observed.

N3

Crystal structures of 1 (two independent molecules), 2, and 3 were determined. Features of the molecular geometry are set out below in terms of the common arylazido moiety, in which R represents an attached or fused pyrimidine ring.

X

Table 1. Geometry of azido group attachment and ring junction

Mol.

C-Nl

la

1. 38 1. 36

lb 2 3

N1-N2 1.24

N2-•N3 (8)

1. 401

1. 35 1.218

1. 16 1. 16 1. 111

1.416

1.255

1. 136

c a - c - n i C x -C-Nl

«

/3(°)

119.1

117 116 117.4

171 168 173.2

115.9

115.6

172.5

125 126

117 114

123.0 124.0

1007

Table 1 (continued) Mol. CA-C-N1-N2 la lb 2 3

- 6

-0.4 -3.7

Cx-C-Nl-N2

C-N1-N2-N3

169 175 179.9 175.8

-179 173 -177.4 -178.6

E(°) 112 -86

-73.4 N.A.

The N^ group of lb makes only distant intermolecular contacts; hence it suffers from high temperature factors and imprecise geometrical parameters. In all the compounds the N^ unit is almost linear (#£170°) and N2-N3 is shorter than N1-N2. The rather sharp bend at N1 (a< 120°) brings N2 into contact with the adjacent hydrogen atom at position A. This contact is relieved by opening the C^-C-Nl angle at the expense of C x ~C-Nl, even when X is as bulky as chlorine. Strain relief via out-of-plane twisting about C-Nl is only of minor importance. Pyrimidine geometry in 2 corresponds closely to that in the similarly substituted metoprine (2). There is a large twist between pyrimidine and phenyl rings as measured by ©, the C6-C5-C11-C2' torsion angle, which is 110° in metoprine and matches this value to within a twofold rotation in 2, while the change to a 6-ethyl substituent in i. permits but does not require an even greater twist. The diaminopyrimidine moieties in all three crystal structures follow the general pattern (3) by forming N4-H...N3 hydrogen bonds around a centre of inversion to make a base pair. The protonated ring N1 and the 2-amino group direct protons towards anions to make further hydrogen bonds. The azido groups enter into van der Waals contacts only. Inhibition data against rat liver DHFR show that it is possible to improve an inhibitor by introducing an azido substituent where it may contact a hydrophobic region of DHFR. Thus K^ values with 95% confidence limits are 1.6 + 0.4 nM for _1 compared with 2.6 + 0.3 for pyrimethamine lacking the azido group,

1008

2.6 + 0.8 for 2, and 0.12 + 0.04 for metoprine. Similarly, 2 inhibits mouse L1210 and E. coli RT500 (form I) DHFR more strongly than the rather feeble inhibitors 2,4-diaminoquinazoline and its 6-nitro- and 6-amino-congeners. Even so, two determinations of K^ for _3 against L1210 DHFR gave 5 1 + 6 and 4 6 + 3 nM. Determinations of pK

and log P (octanol-water) for 1 yielded 7.19 — a and 2.94 respectively, compared with 7.54 and 1.25 for the more basic and polar amine product of reduction. In antitumour screening tests 1 proved to be active. This compound is presently in Phase I clinical trial as an antitumour agent. Preliminary results indicate that f°r elimination is 32-43 h. As intended, this is shorter than it is for many other lipophilic antifolates.

Acknowledgements We thank the Cancer Research Campaign for support and the Science and Engineering Research Council for studentships No. 82314925 and 83700621. Computations were carried out at the University of Manchester Regional Computer Centre.

References 1. Cavallito, J.C., C.A. Nichol, W.D. Brenckman Jr., R.L. DeAngelis, D.R. Stickney, W.S. Simmons and C.W. Sigel. 1978. Drug Metabolism and Disposition 6, 329-337. 2. Cody, V.. 1983. Cancer Biochem. Biophys. 6, 173-177. 3. Schwalbe, C.H., V. Cody. 1983. In: Chemistry and Biology of Pteridines (J.A. Blair, ed.). Walter de Gruyter, Berlin . New York. pp. 511-515.

SUMMARY NOVEL ANTIFOLATES Robert C. Jackson Warner-Lambert/Parke-Davis Pharmaceutical Research Ann Arbor, Michigan 48105

The first fifteen years in the history of antifolates as chemotherapeutic agents were highly successful, leading to the anticancer agent, methotrexate, the antimalarial pyrimethamine, and the antibacterial, trimethoprim.

After these early successes,

synthetic efforts continued unabated but for over two decades no new antifolates were introduced into clinical practice and the resulting disappointment and disillusion caused the impression in some quarters that folate analogues were no longer a fruitful area of medicinal chemistry.

In the 1980s this situation has been

transformed, with four new and active agents introduced into clinical trial, and others in advanced preclinical evaluation. The development of 10-ethyl-10-deazaaminopterin

(10-EDAM) repre-

sents the end result of a process of optimization of selective transport and polyglutamylation of drug by tumor cells relative to normal tissues.

10-EDAM is thus a classical antifolate in

which the features believed to confer antitumor selectivity on methotrexate have been maximized.

Meanwhile, two nonclassical

antifolates have been developed to clinical trial.

These com-

pounds, trimetrexate (CI-898) and piritrexim (BW301U) are not transported by the methotrexate carrier and cannot be polyglutamylated.

Despite this, these new nonclassical antifolates are show-

ing promising clinical activity.

10-Propargyl-5,8-dideazafolate

(CB3717) is the first clinically active antifolate directed at a target other than dihydrofolate reductase (DHFR).

CB3717 has

demonstrated that thymidylate synthetase (TS) is an effective target for anticancer agents.

Among compounds still in preclinical

development, 5,10-dideazatetrahydrofolate

(DDATHF) is particularly

interesting in showing excellent antitumor activity in murine systems, though not inhibiting either DHFR or TS.

Beardsley et

al. (this symposium) have demonstrated that GAR transformylase is the primary site of action of DDATHF.

Chemistry and Biology of Reridines 1986 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed In Germany

1010

The papers presented in session 26 provide clear evidence of this reawakening of interest in the development of novel antifolates. The work of Manteuffel-Cymborowska et al. was one of two presentations showing formation of polyglutamate derivatives of CB3717. Clearly polyglutamylation has implications for drug retention and affinity of CB3717 for TS.

As yet, the extent to which polyglut-

amylation contributes to antitumor selectivity of CB3717 remains unclear.

The presentation of Hynes et al. also deals with class-

ical quinazoline analogues of folic acid, including isofolic acid analogues and examines activity of these compounds as substrates for folypolyglutamate synthetase.

All the compounds tested were

substrates, and some were excellent substrates.

The extent of

polyglutamylation, i.e. the number of glutamate residues added to quinazolines, in comparison with pteridines, is still unknown. Piper et al. describe the synthesis and biological testing of 5substituted 5-deaza analogues of classical folate antagonists. These compounds are effective DHFR inhibitors, and some members of the series have good activity against the E0771 murine tumor. The series contains compounds with 10-deaza structures, including the 5-methyl analogue of DDTHF. synthesis and properties of aminopterin.

DeGraw and colleagues report the

5,10-methylenetetrahydro-8,10-dideaza-

This was a rather weak inhibitor of growth of L1210

cells, but the possibility remains that, like DDTHF, it may be more active in solid tumors.

Bryant et al. provide evidence of

the continuing interest in lipophilic DHFR inhibitors in a discussion of azido-substituted antifols.

Their concept is that

these compounds may provide the desirable pharmacokinetic properties of lipophilic compounds, but that within the cell the azido group will hydrolyze to a more polar amine, resulting in good cellular drug retention.

One compound from the series,

MZPES, is now in phase I clinical trial as an antitumor agent. The recent spate of new compounds and new ideas in antifolate development probably owes its success not only to the persistence of several strong groups of medicinal chemists, but also to the feedback from recent advances in folate biochemistry.

The

stimulation that we have all received from this continuing series of symposia on the Chemistry and Biology of Pteridines has surely played a part in this process.

AUTHOR INDEX

Akiba, K. 287 Akino, M. 223,275 Al-Haddad, D. 509 945,981 Allegra, C.J. Amoroso, D. 681 Anderson, J.M. 327 Andersson, K.K. 201 Andondonskaja-Renz, B. 251,431 Antoulas, S. 95 Appleman, J.R. 769,803 Ardizzoni, A. 681 Armstrong, R.A. 327 Aschhoff, H.J. 339 Avery, T.L. 9 63 Awad, W.M. 913 Ayling, J.E. 51,391 Baccanari, D.P. 789,811,823 Bacher, A. 103,227 Bailey, S.W. 51,391 Bairnsfather, L. 489 Banks, S.D. 835 Baram, J. 945,981 Bartunik, H.D. 103 Beardsley, G.P. 61,953 Beaulieu, D. 843 Bel, Y. 335 Bell, C. 351 Benkovic, S.J. 13 Benz-Lemoine, E. 419 Berteiii, G. 681 Bertino, J.R. 793 69 Besserer, J.A. Biehl, K. 257 Bigham, E.C. 111,835 Blair, J.A. 327,509 Blakley, R.L. 769,803 Blecha, H.G. 427 Bliss, E.A. 1005 Bloom, L.M. 13 Bognar, A. 733 Böhm, P. 33 9 Boritzki, T.J. 69 Botez, M.I. 527 Bottiglieri, T. 523 Bowers, S.W. 219 Boyle, P.H. 91 Bracco, G. 407 Broom, A.D. 631 Brown, G.M. 125,183,295 Bryant, P.K. 1005 Bujard, H. 839 Burchall, J.J. 789 Burgert, S.L. 555

Calvert, A.H. 645,675 Carl, G.F. 495 Carney, M.W.P. 523 Chabner, B.A. 945,981 Chan, P.Y. 929 Chanarin, I. 709 539 Chandler, C.J. Chao, J.Y. 377 Chary, T.K.N. 523 Chen, J.T. 13 Chen, T.B.R.A. 687 Chiodo, L.A. 215 Choe, H. 905 Chow, C.W. 351 Chu,, F.K. >~nu r . j\ . 613 D ±J Cìchowicz, tiowicz, D.J. 9 925, 2 E 997 Ciesla, J. 651,663 Clow, C.L. 403 Cody, V. 799,851,969 Colby, J. 1005 Collins, T.D. 489 Conlon, R.M. 475 Conte, P.F. 681 Cook, R.J. 593,893 481 Cooper, B.A. Cossins, E.A. 741,929 Cotton, R.G.H. 231,351,359, 407 Courtney, G. 513 Courtney, L.F. 13 Coward, J.K. 861 Cremer-Bartels, G. 279 Cuello, A.C. 351 Curtius, H.C. 141,299,399 Danenberg, P.V. 985 Darcy-Vri1Ion, B. 579 Daubner, S.C. 13 Davidson, N. 937 Davis, M.D. 363 Deacon, R. 709 DeFrank, T.J. 977 Delcamp, T.J. 807,815,819,959 Delcroix, M. 239 Dennis, T. 887 Dhondt, J.-L. 239,315,385 Di Cecco, R. 933 Dias-Selassie, C. 959 Dierich, M.P. 427 Dillard, S.B. 391 Dix, T.A. 13 Doig, M.T. 973 Dracon, M. 315 Dube, S.K. 793 Duch, D.S. 151,219,283,377

1012

Dunham, W.R.

917

Edelstein, M.P. 377 Eisenga, B.H. 489 Eilwart, J. 209 Emmerich, B. 2 05 Eto, I. 447 243 Evers, M. Farriaux, J.P. 239 Ferone, R. 737 Ferré, J. 107,115,309,335 Fierke, C.A. 13 Finch, R.A. 963 Fitzhugh, A.L. 639 Fiatmark, T. 201 Fletcher, S.R. 61 Fodor, S. 639 Fonrobert, P. 65 Forsch, R.A. 985 Forzy, G. 239 Frasca, V. 697,917 Freisheim, J.H. 571,575,807, 815,819,959,985 Frisius, H. 411 Fry, D.W. 69 Fuchs, D. 263,427,443 Fusco, V. 681 547,847,861,967 Galivan, J. 347 Galloway, M P. 45,671,989 Gaumont, Y. 893 Gettins, P. 299 Ghisla, S. 897 Ghitis, J. 543 Gier, J. de 843 Goldberg, H 85 Gready, J.E Green, J. 901 Griffin, R.J 1005 Grindey, G.B. 953 993 Grzelakowska-Sztabert, B. Guardamagna, 0. 399,407 Guilhot, F. 419 Gulisano, M. 681 Guynn, R.W. 257 Haan, E. 351 Haavik, J. 201 Halsted, C.H. 539 Hamon, C.G.B. 327,509 Hanlon, M. 737 Hansch, C. 959 Hansen, S.I. 603,607 Harpring, K.M. 571 Harrap, K.R. 675 Harrington, P.J. 61 Hase, Y. 247 Hasegawa, H. 369

Hasler, T. 299,319,399 Hausen, A. 263,427 Hayte, J.M. 239,315 Hengster, P. 427 Hilhorst, E. 687 Hilton, J.G. 481 Hinterhuber, H. 427 Hochuli, E. 839 Hoier-Madsen, M. 607 Holm, J. 603,607 Hoppner, K. 531 Hörne, D.W. 559 395 Howe Iis, D.W. Huber, J.F.K. 263 Huber, R. 103 Hung, J. 69 Hunt, D. 737 31 Hutzenlaub, W. Hyland, K. 395 Hynes, J.B. 57,121,997 Ichiyama, A. 3 69 Inoue, K. 517 Isshiki, G. 247 Iwai, K. 517 Jackman, A.L. 645,675 Jackson, R. 1009 Jacobson, K.B. 107,115 Jansen, G. 543 Jaye, D.L. 235 Jencks, D.A. 697,921 Jennings, I. G. 351,359,407 Joannon, P. 843 Jones, T.R. 675 Joshi, G. 887 Joyner, S.S. 811 Kaiman, T.I. 583,763,985 Kaminska, B. 993 Kang, Y. 31 Kapatos, G. 215 Kaplan, P. 403 Katoh, S. 291 Kaufman, S 169,185,351,363, 639,989 Keating, J.M 905 Kedzierska, B. 663,667 91 Kelly, M.F. 373 Keiner, K.L. 571 Kempton, R.J. 339 Kersten, H. 205 Kersten, W. 847 Kerwar, S. 485,973 Kesavan, V. 691 Keyomarsi, K. Khwaja, T. 959 Kisliuk, R.L. 45,655,671,743, 989

1013 Koch, M.A. 415 Kocher, H.P. 839 Kohashi, M. 517 571 Kohrs, F. Kompis, I. 83 9 Kozloff, L.M. 757 Krajewski, K.J. 257 Krause, H. 279 Kraut, J. 789 Krumdieck, C.L. 447 Kuskinsky, R. 351 Kulikowski, T. 667 Kulinski, R.L. 803 Kumar, P. 989 Kumari, J. 475 Kunze, R. 411,415 Küster, T. 305 Rüther, G. 251 Kwee, S. 73 Laberge, C. 403 Ladenstein, R. 103 Lampi, B. 531 Lange, W. 415 Lapp, W. 4 81 Largilliere, C. 239 Laundy, M. 523 Leeming, R.J. 475 Lehmann, H. 399 Lelievre, G 315 Leupold, D. 69,399 Leskopf, W. 31 Levine, R.A. 347,373,381 Lewis, G.P. 909 Lowe, K. 925 Lumb, M. 709 Lura, R. 887 Machnicka, B. 651 MacKenzie, R.E. 767,901 Maley, F. 613 Maley, G.F. 613 Mandelbaum-Shavit, F. 597,855 Mangum, J.H. 913 Manteuffel-Cymborowska, M. 993 Masada, M. 223,275 Matasovic, A. 305 Matsumoto, J. 275 Matsuura, S. 77,81,223 Matthews, D.A 789 Matthews, K.D, 697 Matthews, R.G, 697,901,917, 921 Maubach, P. 205 Mayer, R.J. 13 McAdam, W.J. 351 McCaleb, G.S. 1001 McGuire, J.J. 729,861 McMartin, K.E, 489

McNulty, H. 513 McPartlin, J.M. 513 Meij, P.F.C. Van der 687 Mensua, J.L. 323 Merrill, D.K. 257 Milman, G. 925 Milstien, S. 169,403 Mohyuddin, F. 243,403 Molloy, A.M. 505 Montgomery, J.A. 1001 Moran, R.G. 645,691,937,953 Morgan, F. 351 Morrison, J.F. 827,831 Mura, P. 419 Murata, S. 77,81 Nagatsu, T, 223 Nair, M.G. 45,989 Nakagoshi, M. 271 Nakanishi, N. 287 Nakata, H. 351 Nayak, V.K. 583 Naylor, E.W. 309 Nazarbaghi, R. 659 Neuberger, G. 227 Newell, D.R. 675 Newton, P.A. 9 63 NextfS, E. 603 Nichol, C.A. 151,219,283,377, 835 Niederwieser, A. 141,305,319, 399 Nimec, Z. 547 Noel, C. 315 Noronha, J.M. 485 O'Connor, D.L. 555 Oatley, S.J. 789 Okano, Y. 247 Okeke, C.C. 57 733,925 Osborne, C. Oura, T. 247 Ozawa, K. 287 Panos, C.H. 57 Parniak, M.A 351,359 Pathak, A. 57 Paton, D.R. 295 Pawelczak, K 675 Pember, S.O. 13 Perry, J. 709 Petersson, L. 201 Pfleiderer, W. 31,115,305 Pheasant, A.E. 267,509 Picciano, M.F. 555 Pike, D.C. 51 Piper, J.R. 729,803,1001 Piriou, A. 419 475 Pollock, A.

1014

399,407 Ponzone, A. Price, E.M. 571,575 Priest, D.G. 479,973 Primus, J.P. 125 Pristupa, Z.B. 933 Pronzato, P. 681 Przybylski, M. 65 Pupons, A. 547 Rabinowitz, J.C. 865 Ramamurthy, B. 45 Ratnam, M. 819 Ratnam, S. 815 Rebandel, H. 671 Reddy, A.R.V. 583 Rehder, M.C. 847 Reibnegger, G. 263,427 Reinhard Jr., J.F. 111,377 Reisenauer, A.M. 551 Reiss, D. 419 Renkel, R. 65 Renner, D. 427 Repetto, L. 681 Reynolds, E.H. 523 Rhee, M.S. 547 Riazzi, B.S. 917 Rijksen, G. 543 Rode, W. 651,663,667 Roessler, H. 427 Rokos, H. 411,415 Rosenberg, I.H. 563,579,587 Rosenblatt, D.S. 713,843 Rosowsky, A. 807,985 Rosso, R. 681 Rupar, C.A. 243 Rzeszotarska, B. 675 Said, H.M. 567 Sams, L.A. 571 Sands, R.H. 917 Santus, R. 99 Sato, J.K. 287,977 Sawada, M. 223 Sawada, Y. 247 Scheibenreiter, S. 305 Schirch, V. 887,891 Schmidt, H. 399 Schneider, M. 103 Schornagel, J.H. 543 Schott, K. 103 Schreiber, C. 897 Schulz, T. 427 Schwalbe, C.H. 1005 Schweitzer, B.I. 763 Schwulera, U. 209 Scott, J.M. 467,505,513 Scrimgeour, K.G. 933 Scriver, C.R. 403 Seihub, J. 563,579 Shane, B. 719,733,925,997

Shannon, K.W. 865 Sherman, A.R. 555 Shih, C.J. 61 Shintaku, H. 247,399 Shugar, D. 667 Sikora, E. 675 323 Silva, F.J. Singer, S. 737 Sirotnak, F.M. 1001 Skiba, W.E. 913 Slieker, L.J. 13 Smith, A.G. 571 Smith, G.K. 111,151,835 Smith, I. 395 Smith, P.L. 575,807 Smith, S.L. 789 Soyka, R. 31 Spiro, T.G. 639 Srimatkandada, S. 793 Stäben, C. 865 Staudenmann, W. 305 Steinberg, S.E. 471 Steinerstauch, P. 299 Stevens, M.F.G. 1005 Stokstad, E.L.R. 717,905 Stone, S.R. 827,831 Strong, B. 887 Strum, W.B. 567 Struppler, A. 251 Stüber, D. 839 Sueoka, T. 291 Sugimoto, T. 77,81,223 Sussman, D. 925 Sutton, P.A. 969 Suyama, I. 247 Switchenko, A.C. 125 Szewczyk, B. 757 Szewczyk, K. 757 Tacquet, A. 315 Taira, K. 13 Takikawa, S. 141,299 Talmadge, K. 839 Tansik, R.L. 823 Tanzer, J. 419 Taylor, E.C. 55,61,953 Taylor, S.M. 937 Then, R.L. 839 Thorndike, J. 655 Toghiyani, T.R. 45 Toone, B.K. 523 Tsuruhara, T. 247 Tsusue, M. 271 Unterweger, B. Vickers, P.J. Volk, R. 227 Vuchich, M.J.

427 933 843

1015 687 Waard, E.R. -de 263,427,443 Wächter, H. 593,893 Wagner, C. 539 Wang, T.T.Y. 713 Watkins, D. 403 Watters, G.V Waxman, S. 897 Webber, S. 235,659 Weibel, E.K. 839 Weir, D.G. 467,505,513 Wells, M.S. 913 Welsh, W.J. 799 Werbel, L.M. 69 Werner, E.R. 263,427 West, D.K. 613 Whitehead, T .R. 865 Whitehead, V •M. 843 Whiteley, J.;M. 659 Wick, M.M. 985 Wiesenfeldt, M. 31

Wilson, E. 467 Winkler, F. 839 Wong, G.S.K. 61 Wong, K.P. 1005 Woo1f, J.H. 283 Wright, J.E. 807 Yamada, S. 287,291 Yamamoto, H. 247 Yang, I.Y. 631 115 Yim, J.J. Zeitler, H.-J. 251 Zheng, Y.C. 959 Zhu, H. 959 Ziegler, I. 209 Zielinski, Z 651, Zimmermann, J. 563 Zubrod, E. 205,335

SUBJECT

INDEX

Only the f i r s t page number of the c o n t r i b u t i o n in which the keyword appears i s given. Absolute c o n f i g u r a t i o n , hydropterins 77

6-A1koxymethyl-5,6,7,8-tetrahydropterins - synthesis 111 - cofactor a c t i v i t y 111

tetra-

Acetamidobenzoylglutamate

467

N(5)-Alkylation

6-Acetyl-7,8-dihydrohomopterin - complex with

107

N-Acetylserotonin

151,169

A c e t y l s e r o t o n i n methyl t r a n s f e r a s e 279 A c t i v e s i t e probes Acyl phosphate ADE 3 gene

631

A l l o s t e r i c model

921

Alpha-hydroxyglutaric acid

719

743

Amino a c i d a n a l y s i s , of p l a n t and human DHFR 815

865

Adenosine deaminase

655

Amino a c i d s , aromatic, metabolism

Adenosylhomocysteine 593 - as source of homocysteine

909

Adenosylmethionine 593 - i n h i b i t i o n of m e t h y l e n e t e t r a h y d r o f o l a t e reductase 697 - as i n h i b i t o r 921 - and methionine s y n t h e t a s e 909 Adrenal medullary cells 373

95

6-AlkyIthiomethy1-5,6,7,8-tetrahydropterins - synthesis 111 - cofactor a c t i v i t y 111

chromaffin

Amino a c i d s , t r a n s p o r t p-Aminobenzoylglutamate

467,513

2-Amino-4-keto-6-methyl-7,8-dihydropyrimidodiazepine 51 2-Amino-4-oxo-6-acetyl-3H,9H-7,8-dihyd r o p y r i m i d o ( 4 , 5 - b ) ( l , 4 ) - d i a z e p i n e (PDA), substrate for drosopterins 295

A f f i n i t y chromatography

977

Aminopterin, 5 - a l k y l - 5 - d e a z a 1001

Affinity

807

4-Aminopteroyl-polyglutamyl-esters

Age

l a b e l i n g , DHFR

AIDS

945,989

Amniotic f i b r o b l a s t s

427

309

Amniotic f l u i d p t e r i n s

399

Alcohol - chronic effects 489 - and f a t t y l i v e r 505 - and f o l a t e d e f i c i e n c y 489 - and f o l a t e e x c r e t i o n 489 - and f o l a t e metabolism 489

A n t i c a n c e r drugs

Alcohol

Anticancer folate i n h i b i t o r s

ingestion

Alcoholism, chronic

Amyotrophic l a t e r a l

467 485

6-A1koxymethyldi h y d r o p t e r i ns 6-Alkoxymethyl-7,7-dimethyl 5,6,7,8-tetrahydropterins - synthesis 111 - cofactor a c t i v i t y 111

analogs 65

6 - A m i n o u r a c i l s , as apoenzyme-substrate complex models 687

279

AICAR t r a n s f o r m y l a s e

335

185

671

Antibodies

351,607,819,977

A n t i f o l a t e drugs

251

993,1005

Antifolate activity 835

sclerosis

Antagonism

799

45

1005

Antifolates 69,729,815,861,945,953, 977,981 - adamantyl851 - arthritis 847

1018 -

biology 967 CB 3717 993 crystallographic analysis 851 5-deaza analogs of c l a s s i c a l 1001 l i p o p h i l i c 851 novel 1009 t r i a z i n e 959

A n t i f o l a t e s , spin-labeled 803 - immobilization of ligands 803 - mobilization of side chains 803 - synthesis

803

Antioxidant

547

Aspirin

239, 315, 385

L-erythro-biopterin

251

Biopterin synthetase deficiency B i o t i n Conjugate

363

107

Brain, folate concentrations

Avidin peroxidase

Breast cancer

613

Baseplate assembly

B. s u b t i l i s

103,227,743

Benzoquinone a c t i v a t i o n

563,567

743

Candida gui 11iermondii

757

Carbamazepine 603

227

495

Carbodiimide a c t i v a t i o n

757

Betaine 913 - effect on methionine synthetase 909

Carbonyl oxide, probe for

BH^

Carboxypeptidase Y

see Tetrahydrobiopterin

769

C a t a l y t i c oxygen reduction

Biogenic amine metabolites

CB 3717

385

305

Biomimetic methylene transfer Biopterin 151,271,305,327,339 - in amniotic f l u i d 239,399 - in chronic uraemia 315 - clearance 315 - deficiency 239,243,247,385 - diurnal v a r i a t i o n 267 - excretion, cocaine 257 - in human brain 223 - l e v e l s , human 247 - in l i v e r of animals 319 - N/B r a t i o in urine 243 - in plasma 475 - in red blood c e l l s 475 - reduced d e r i v a t i v e s 235

687

583

737

B i o a v a i l a b i l i t y , of tetrahydro and dihydropterins 835 Biolumazine

51

Carboxypeptidase G, i n h i b i t i o n C a t a l y t i c mechanism

531

495

539,563

Brush border membrane

1005

419

681,945,981

Brush border

607

Bacillus subtilis

Bioassay

419

- treatments, neopterin v a r i a t i o n s

531

Autocatalytic s p l i c i n g

385

607

Bone marrow transplantation

847

Azido compounds

- in urine

Bond Elut, C18

Anti-PAH antibody Arthritis

- in serum 239, 315 - s y n t h e s i s , defective 403 - s y n t h e s i s , inborn error of metabolism 239 - urinary excretion 267

73

Catecholamine synthesis 201,373 993

CB 3717 triglutamate, i n h i b i t i o n of thymidylate synthase 763 CCRF-CEM c e l l s

729,953

Cell mediated immunity

427

Cerebrospinal f l u i d 251,385,395 - biopterin p r o f i l e s 395 - HVA and 5HIAA concentrations 395 5-CH,-H.PteGlnc, i n h i b i t o r of glycine N-meïhyTtransferase 593 Chinese hamster ovary c e l l s Choline turnover Chorionic v i l l i Chromaffin c e l l s

593 309 373

843

1019

Chronic alcoholism

Cytochrome b

485

C h r o n i c ethanol e f f e c t s

489

C4a-hydroxydehydratase

Cytotoxity

51

Circadian rhythm - of Cortisol 411 - of n e o p t e r i n 411 - of T - L y m p h o z y t e s 411 315

- neopterin

315

Cloning

DAEP

DAMP

733

681 calcu-

Coba 1 amin 481,713 - inactivation 709 - in m e t h i o n i n e s y n t h a s e Cocaine a b u s e , p t e r i n Colour dimorphism Conformational

DAPP

969

hydro-

synthase

Crithida fasiculata

185

polyglutamates

989

5-Deazaantifolates,

5-substituted

1001

45

11-Deazahomofolate

45

5-Deaza-5,6,7,8-tetrahydrofolicacid 953 893

Depression Desilylation

607

structures

DHFR

969,

523

C18 s i l i c a c a r t r i d g e s C.-Tetrahydrofolate 865,887

107

synthase

Cumene hydroperoxide Cyclic AMP, analogs

547 283 73

Cystathionine-gamma-synthase 743

305

2'-Deoxysepialumazine 2'-Deoxysepiapterin

757

Cyclic voltammetry

45

2'-Deoxybiolumazine

Copper,in methionine 697

Cross-reactivity

61

10-Deazaaminopterin

Demethylation

823

799

851

10-Deazafolate

271

comparison

Cooperati vity

697

799

851

10-Deazaaminopterin

excretion

C o n j u g a s e (y-glutamyl lase) 583,933 - inhibition 583

CSF folate

799

851

D D A T H F and a n a l o g s

Crossi inking

799

851

D A P P , as DHFR i n h i b i t o r DCXMP

327

Crystal 1005

851

D A M P , as DHFR inhibitor 865

C N D O / 2 M o l e c u l a r orbital lations 799 CNS

861,953

D A H P , as DHFR i n h i b i t o r

C l o s t r i d i u m acidi-urici CMF

339

D A E P , as DHFR i n h i b i t o r DAHP

Clearance - biopterin

339

Cytochromes

305 305

523 91

see D i h y d r o f o l a t e

reductase

2,4-Diamino-5-(l-adamantyl)-6-ethylp y r i m i d i n e (DAEP) 851 2,4-Diamino-5-(1-adamantyl)-6-methyl p y r i m i d i n e (DAMP) 851 2,4-Diami n o - 5 - ( 1 - a d a m a n t y l ) - 6 - p r o p y l p y r i m i d i n e (DAPP) 851 2,4-Diami n o - 5 - ( 1 - a d a m a n t y l ) (DAHP) 851

pyrimidine

2,4-Di ami n o b e n z y 1 p y r i mi di nes 2,4-Diamino-5-t-butyl-6-methyl ine (DTMP) 851

823 pyrimid-

1020 2,4-Diamino-5-cyclohexyl-6p y r i m i d i n e (DCXMP) 851

methyl

2,4-Diamino-5,8-dideazapterins, s y n t h e s i s and p r o p e r t i e s 57 4,6-Diamino-l,2-dihydro-2,2-dimethyl-l-(X-phenyl) triazines 959 2,4-Diamino-6-hydroxy 231

pyrimidine

2,4-Diaminoquinazolines,synthesis and p r o p e r t i e s 57 2,4-Diamino-6-pteri dinecarboxaldehyde 99 - p h o t o s e n s i t i z a t i o n by 99 - t r i p l e t s t a t e 99 Diaminopyrimidines

69

2,4-Diaminopyrimidines, 1ipophilie, as DHFR i n h i b i t o r s 799 Dibutyryl

c y c l i c AMP

5,8-Dideazafolates 5,8-Dideazafolic

287 997

acid

645

5,10-Dideaza-5,6,7,8-tetrahydrof o l i c acid 953 - and a n a l o g s 61 - asparate analog 953 - diastereomers 953 Dietary

iron

Differential

Differentiation

447

275

-

2,6-Difluorobenzonitrile, reactions 57 7,8-Dihydrobiolumazine Dihydrobiopterin

305

151,835

7,8-Dihydrobiopterin Dihydrobiopterin,

305

quinonoid

Dihydrobiopterin synthetase deficiency 243,399

-

363 cleavage

• -

81 (DHBS)

7,8-Dihydro-2'-deoxybiolumazine 305 Dihydrofolate 769,789,831,835, 945 - determination of 973 - i n t r a c e l l u l a r accumulation 981 - r e s p o n s e to m e t h o t r e x a t e 973 Dihydrofolate reductase 13,151,395, 645,671,719,729,757,769,803,819,843, 851,861

-

affinity labeling 807 a l t e r a t i o n o f gene 793 amino a c i d a n a l y s i s 815 antibodies 819 antibody c r o s s - r e a c t i v i t y 819 binding of i n h i b i t o r s 769 bovine 835,839 chicken l i v e r 831 complex d i s s o c i a t i o n c o n s t a n t s 803 crystallization 769,839 crystallography 769 deuterium isotope e f f e c t s 827 dihydrofolate 769,831 dihydropyrirnidodiazepine substrate for 51 E.coli 811,827,835 e n z y m e - N A D P H - i n h i b i t o r complex 823 folate 769,831 f o l a t e complex 811 human 793,839,937,977 inhibition 69,799,815,855,945,959,989 i n h i b i t i o n by MTX a n a l o g s 985 inhibition constants 769 inhibitor binding 823 inhibitor dissociation constants 823 i s o m e r i z a t i o n o f complexes 769 isozymes 811 k i n e t i c mechanism 769 k i n e t i c parameters 827,831 kinetic properties 815 l a c k i n g , C h i n e s e hamster o v a r y c e l l s 843 l i g a n d complexes 819 1 i g a n d - i n d u c e d c o n f o r m a t i o n a l change 819 mapping o f a n t i g e n i c y 819 mechanism 769,827 methotrexate 769 mouse S - 1 8 0 839 m u l t i p l e enzyme forms 811 n e i s s e r i a gonorrhoea 823 NMR 769 o l i g o n u c l e o t i d e - d i r e c t e d mutagenesis 769 pH e f f e c t s 827,831 photoaffinity labeling 575 pK a v a l u e s 827,831 plant 815 p l a s m i d e encoded 789 purification 811,815 radioimmunoassay 818 rat 839 reactive groups 807 r e c e n t advances 769 recombinant 839 sheep l i v e r 989 s i t e - d i r e c t e d mutagenesis 793 soybean 815

1021 -

stopped-flow f1uorimetry 2,8 A structure 789 substrates 799 substrate s p e c i f i t y 831 X-ray d i f f r a c t i o n 769 X-ray structure 839

6,7-Dimethylhydropterin, quinonoid

769

6,7-Dimethylpterin

6,7-Dimethyl-8-ribityllumazine

103,227

6,7-Dimethyl-5,6,7,8-tetrahydropterin 91

Dihydrofolate s y n t h e t a s e - f o l y l p o l y glutamate synthetase, E. c o l i 737

6(1',2'-Dioxopropyl)tetrahydropterin 151

D i h y d r o f o l i c acid

Disorders, neurological

- substrate for DHFR

799

7,8-Dihydro-6-hydroxylumazine Dihydromethanopterin

305

743

6,6-Disubstituted tetrahydropterins DNA sequence

Dihydroneopterin trisphosphate 151,299 - d e r i v a t i v e s 115 - t r i s effect 115

- s y n t h e s i s , modulation

Dihydroorotate

Drosophila melanogaster

Domains

DOPA formation Dopamine

Down's syndrome

Dihydropterins - b i o a v a i l a b i l i t y 835 - quinoid 6 , 6 - d i s u b s t i t u t e d 391 - as substrate for drosopterins 295

E. c o l i DHFR

743, 989

327 323,335

- nonenzymatic synthesis Drug addicts DTMP

295

427

851

DTMP, as DHFR i n h i b i t o r Dynospheres

799

603

Dyspropterin reductase

275

Dyspropterin synthetase E. c o l i

275

743 811

Electrical excitability

215

Electron spin resonance

803

Enzyme a c t i v i t y

959

Enzyme amplification Enzyme conformation

733 769

see i n h i b i t o r s

Enzyme Linked Immuno Sorbent Assay (ELISA) 607

291 835

Dimethylglycine dehydrogenase

347

215

125

Enzyme i n h i b i t o r s

305

6,7-Dimethyl dihydropterin

377

373

Dihydropteridine reductase 185,287, 309,315,391 - a c t i v i t y in animal l i v e r 319 - biopterine p r o f i l e s in CSF 395 - deficiency 385,395,399,407 - homovanillic acid and 5-hydroxyindole acetic acid concent r a t i o n s 395 - human mutant 407 - hyperphenylalaninaemia 395 - quinoid dihydropyrimidodiazepine substrate for 51 - substrates for 111

Dihydropteroylpentaglutamate

937

865

Drosopterin

Dihydropteroylhexaglutamate 757

865

DNA transfection

Dopamine neurons

509

a-Diketo reductase

391

D i s s o c i a t i o n constants 769 - of DHFR complexes 803

7,8-Dihydroneopterin - circadian rhythm 411 - influenced of s t r e s s 411 - in LAV/HTLV-III i n f e c t i o n 411 - in renal i n s u f f i c i e n c y 411 Dihydroneopterin/Neopterin-ratio 411

7,8-Dihydroxanthopterin

81

91

893

Enzyme regulation, k i n e t i c s

921

391

1022 Enzymes - monofunctional 865 - multifunctional 865 - trifunctional 901 Enzymology

953

Epileptics

527

EPR spectroscopy

201,917

Equilibrium d i a l y s i s

603,823

Erythrocytes - folate quantitation 603 - 6-pyruvoyltetrahydropterin synthase a c t i v i t y 399 - sepiapterin reductase a c t i v i t y 399 Erythrophore

275

Erythrophoroma cell

275

10-Ethyl-10-deazaaminopterin 855

45,

10-Ethyl-10-deazaaminopterin glutamates 989

poly-

N(5)-Ethyl-heptaacetyl-5,6,7,8-tetrahydro-D-neopterin 95

- urinary excretion Folate absorption Folate analogs

Fluorescence, tryptophan

o-Fluorobenzoni t r i l e s , preparati on and reaction 57 Dl-threo-4-Fluoroglutamic acid Fluoropyrimidines

729,997

743

691

681,691

Folate see also f o l i c acid 495, 555,769,831 - absorption and transport 587 - active transport 567 - bioavailability 531 - chemistry 55 - CSF 523 - d i e t a r y , digestion 539 - DHFR folate complex 811 - effect of anticonvulsant t r e a t ment 495 - excretion, and alcohol 489 - formylation 709 - human 523 - i n t e s t i n a l absorption 551,579 - i n s t e s t i n a l transport 563,567 - intracellular 811,843,981

953

567

Folate-binding proteins 551,567 - immobilized 603 - i n s o l u b i l i z e d 603 - in milk 607 - in serum 607 - soluble 603 Folate catabolism 467,513 - effect of xanthopterin and a l l o p u r i n o l 509 - in hamster 509 Folate deficiency

467,505,523

489

Folate depletion

201

485,489 485,709

Folate antimetabolites

- and alcohol

893

5-F1uorouraci1

l e v e l s in e p i l e p t i c s 527 l i v e r 471,531 metabolism, and alcohol 489 microbiological assay 843 quantitation 603 red c e l l 523 reduced 843 regulation 925 renal clearance 489 in serum 523 in t i s s u e s and c e l l s 479

Folate binders

ESR spectroscopy, methionine synthase 697

Flavoenzyme

-

843

Folate homeostasis 925 Folate i n h i b i t o r s , theoretical 799

studies

Folate metabolic mutant c e l l s , complementation of 937 Folate monoglutamates 517 - detection and determination - serum l e v e l s 517 Folate oligoglutamate 743

517

transpeptidase

Folate polyglutamates, i n t e s t i n a l sorption 579

ab-

Folate requiring enzymes, mechanism of reaction 13 Folate rescue, in lymphocytes Folate transpeptidase

481

933

F o l i c acid see also folate 843,905 - storage in red blood c e l l s 471 - structure 969 - substrate for DHFR 799

1023 - supply 471 - transport 551,559

Gene cloning

Folinic acid

Glutamate

681,691

Folylpolyglutamate dase 757

carboxypepti-

folylpolyglutamate hydrolase

933

Folylpolyglutamate reductase

861

Folylpolyglutamates 811,925,933 - E. coli, analysis 737 - E. coli, synthesis 737 - kinetic constants 719 - synthesis 719,741,925 Folylpolyglutamate synthetase 729, 733,743,757,763,925,997 - cloning of 937 - corynebacterium 719 - E. coli 719 - endogenous inhibitor of 929 - formation of di and tri glutamates 929 - human 937 - kinetic constants 719 - L. casei 719 - mechanism of action 719 - pig liver 719 Folylpoiy-a-glutamate E. coli 737 Formaldehyde Formate

synthetase,

505,743

Goldfish

275

Graft-versus host disease 419

709

151 603,865

10-Formyltetrahydrofolate 887,945

865,

10-Formyltetrahydrofolate: oxidoreductase 905

NADP

10-Formyltetrahydrofolate tase 865,887

GTP cyclohydrolase 219,275,283,315 - activity, effect of 3-Hydroxykynurenine 323 - deficiency 385 - fast assay procedure 415 - hormonal regulation 219 - in LAV/HTLV-III infection 415 - in monocytes, macrophages 415 GTP cyclohydrolase I 151 - activity in animal liver - in human brain 223 655

synthe-

283

743

Gamma-fluoromethotrexate

319

655

reconstitution

861

HCT8 cell line 419 - development of MTX-resistant cell line 793 - DHFR cDNA cloning 793 - DHFR gene amplication 793 - southern analysis 793 Hepatic polyglutamate synthesis

10-Formyltetrahydropteroy1glutamate 517

Gamma-diglutamate

(G.V.H.D.)

Growth inhibition 953,959 - and hydrophobicity 959

H35 cells

953

5-Formyltetrahydrofolate

Forskolin

Glycine N-methyltransferase - human 593 - rat 593

Haemopoietic

Formylation, folate

Formylpterin

trans-

905

865

Formyl-GAR

547

Glycine amide ribonucleotide formylase 945

Guanosine

Formiminoglutamic acid

205,733,925

719

Glutathione

Guanase

743

865,905

Formylase

865

Gene expression

861

Hepatocytes

547,559

Hepatoma cells Histidine HL-60 cells

485

743

905 897

Homocysteine - formation from adenosylhomocysteine 909 - stabilisation of methyltetrahydrofolate 909 - as substrate for methionine synthetase 909

1024

Homocystinuria

6(1'-Hydroxy-2'-oxopropyl)tetrahydropterin 151

713

Homovanillic acid

385

HPLC 467 - biopterins 395 - of cerebrospinal f l u i d , p t e r i d i n e s 251 - chiral 861 - d o p a m i n e and S e r o t o n i n e m e t a b o lites 395 - e l e c t r o c h e m i c a l and f l u o r e s c e n c e detections 395 - 5 - f o r m y 1 t e t r a h y d r o p t e r o y 1 gl u t a mate 517 - 10-formyltetrahydropteroylglutamate 517 - 5-Methyltetrahydropteroylglutamate 517 - neopterin 263 - pABGlu, ApABGlu 513 - p t e r i d i n e s X, X I , X2, X3 107, 115 - pteroylglutamate 517 - tetrahydropterins 835 - tetrahydropteroylglutamate 517 - u r i n e , N / B r a t i o and BH4 243 - urine, pteridines 251 H2PTeGlu5

743 virus

299

H y d r i d e transfer

901

Hydrodynamic voltamogram, d r o b i o p t e r i n in CSF 395 Hydrogénation

427

tetrahy-

91

H y d r o g e n bonding

969

Hydrophobic pocket

851

7-Hydroxybiopterin

271

811

219

Hypothyroidism

905

1 3 I m i d a z o l i n e s , N ,N - u n s y m m e t r i c a l l y disubstituted 687 I m m o b i l i z a t i o n of l i g a n d s Immunity, cell m e d i a t e d Immunoglobulin, Indoles

5-Hydroxyindoleacetic 3-Hydroxykynurenine radicals

6-Hydroxylumazine

acid

385

323 73 305

7-Hydroxymethotrexate - antileukemic activity 963 - e f f e c t on m e t h o t r e x a t e 963 - host t o x i c i t y 963 6-Hydroxymethyl pterin - f o r m a t i o n in T cells 209

803 427

anti-FBP, rabbit

607

169

Infections

419

Inhibition, of DHFR

959

Inhibition c o n s t a n t s , DHFR

769

839

Inhibitors 659,663,675,729,953 - active a g a i n s t F P G S ' s 929 - binding to DHFR 769 - endogenous 929 Interleukin 2 - internalization - radio i o d i n a t e d - signal function

209 209 209

transmission

Intestinal

209

a b s o r p t i o n , folate

551

613

Iron 555,893 - in m e t h i o n i n e

synthase

Iron a b s o r p t i o n

743

231

905

Hypophysectomy

Intron

8-Hydroxy-7-dimethyl-5-deazaflavin

Hydroxyl

- pterin administration Hyperthyroidism

- signal

Hydrophobic chromatography

651

Hyperphenylalaninemi a 239,385,395 - m o u s e model 231 - newborn s c r e e n i n g for 243,403

Inhibitor d e s i g n

Human immunodeficiency H u m a n liver

Hymenolepsis diminuta

697

363

Iron c o n t e n t , of p h e n y l a l a n i n e lase 363 Iron protein

201

Iron s u p p l e m e n t

363

Isodrosopterin, nonenzymatic 295 Isomerase

hydroxy-

synthesis

291

I s o m e r i z a t i o n , of DHFR c o m p l e x e s Isoxanthopterin

125,251,271,305

769

1025 K562 c e l l s

729

Mesencephalon

Kidney - allograft 315 - b i o p t e r i n metabolism Lactate

315

Methanobacterium 743

339

Methanopterin

L - ( + ) - l a c t a t e cytochrom c o x i d o reductase 339 Lactation

555

Lactobacillus casei 6-Lactoyl 291

659,743

tetrahydropterin

Lactoyl-tetrahydropterin 151

99

427

L1210 c e l l s

575,663,763,953,959

- permeabi1 i zed LDH-isoenzymes

763 339

865

L i g h t melanotonin Liver

141,151,

synthase

Laser f l a s h s p e c t r o s c o p y LAV/HTLV-III

Leucovorin

279

913

Liver folate

471,531

- concentrations L1210 leukemia

495 963

L1210/R71 c e l l s Lumazines

959

305

Lumazine synthase Lymphocytes - human 231 - mouse 231 - stimulated

MCF-7 c e l l s

103,227

Mean c o r p u s c u l a r volume

Membrane

Methionine synthase 481,709 - activation 697 - cobalamin-dependent 917 - from E. c o l i 697,917 - EPR s p e c t r o s c o p y 917 - i n a c t i v a t i o n by n i t r o u s oxide - from p i g l i v e r 697 Methionine s y n t h e t a s e - assay requirements - blanks f o r 909

275 169

567

2-Mercaptomethylglutaric acid 583 - design, synthesis 583 - carboxypeptidase Gi i n h i b i t i o n 583 - conjugase i n h i b i t i o n 583

917

505 909

Methotrexate 543,547,597,681,729,769, 823,839,847,855,861,897,933,945,977 - 5-alkyl-5-deaza analogs 1001 - as a n t i f o l a t e 981 - as DHFR i n h i b i t o r 799 - e f f e c t s on c e l l growth 973 - e f f e c t s on reduced f o l a t e s 973 - i n t e r a c t i o n , 7-hydroxy-MTX 963 - membrane b i n d i n g components in L1210 cells 575 - p h o t o a f f i n i t y analogues 575 - in plasma 475 - in red blood eel I s 475 - s t r e t c h e d analogs 985 - s t r u c t u r a l analogue 807 - structure 969 571 4-fluoroglu659

Methotrexate p o l y g l u t a m a t e s - formation 843 475

865

Methionine 505,713 - biosynthesis 697,717,913

Methotrexate d e r i v a t i v e s

547

855,981

Melanophore

743

5,10-Methenyl-THF c y c l o h y d r o l a s e

Methotrexate d e r i v a t i v e of tamic a c i d 743

481

syn-

thermoautotrophicum

Methotrexate analogues

Malondialdehyd

Melatonin

215

Metabolism, inborn e r r o r , b i o p t e r i n thesis 239,385

475,945,989

Methotrexate-y-polyglutamates - purity 65 - synthesis

65

65

Methotrexate r e s i s t a n c e

69,543

Methotrexate-sepharose

839

Methotrexate t r a n s p o r t

543

5-Methyl-5-deazaaminopterin 10-Methyl-10-deaza-aminopterin

1001 855

1026 5-Methyl-5-deazamethotrexate

1001

5-Methyl-5,10-dideazaaminopterin 1001

Molecular packing Monapterin

Multifunctional enzymes Multisubstrate i n h i b i t o r

5-Methyltetrahydrofolate 713,945

NAOPH

81

Methyltetrahydrofolate-homocysteine methyl transferase 697 5-Methyltetrahydrofolate-Homocysteine transmethylase 743 acid,

6-Methy1 -tetrahydropteri n homolog 51 5-Methyltetrahydropteroylglutamate 517 6-Methyl-tetrahydropyrimidodiazepine 51

Milk

555

593 865

151

N e i s s e r i a gonorrhoea DHFR

597,603,

913

287

NADH-specific dihydropteridine reductase 287

505

6-Methylhydropterin, quinonoid

Methyl trap hypothesis

631

NADH-dihydropteridine reductase

687

Micrococcus aerogenes

369 865

dehydro-

Methyltetrahydrofolate, s t a b i 1 i zation by homocysteine 909

Methyl transferase

865

Mouse mastocytoma, P-815

Methylenetetrahydrofolate reductase 395,697,905 - a l 1 o s t e r i c model 921 - Clostridium formicoaceticum 697 - i n h i b i t i o n by AdoMet 697 - pig l i v e r 697 - regulation by AdoMet and NADPH 921

5-Methyltetrahydrofolic transport 559

251

Monofunctional enzymes

5,10-Methylenetetrahydrofolate 691,945 - models 687 - substrate for FPGS 929

Methyl group wastage

799

969

Monocytes/macrophages - a c t i v a t i o n of 415

Methylenetetrahydrofolate 743 - determination of 973 - response to methotrexate 973

Methylene transfer

547

MM2p F o r c e - f i e l d c a l c u l a t i o n s

835

Methylenetetrahydrofolate genase 865,901

865

Mitoxanthrone

5-Methyl-5,10-dideaza-5,6,7,8t e t r a h y d r o f o l i c acid 1001 6-Methyl dihydropterin

Mitochondria

823

Neopterin 151,271,419,427 - in amniotic f l u i d 239,399 - in animal l i v e r 319 - biopterin r a t i o 239,315,385 - in chronic uraemia 315 - circadian rhythm 411 - determination 263 - diurnal v a r i a t i o n 267 - excretion, cocaine 257 - formation in T c e l l s 209 - in human brain 223 - influence of c o r t i c o t r o p i n r e l e a s i n g hormone 411 - influence of s t r e s s 411 - in LAV/HTLV-III i n f e c t i o n 411 - in LAV/HTLV-111 p o s i t i v e patients 415 - N/B r a t i o in urine 243 - p r o l i f e r a t i o n of lymphocytes 415 - radioimmunoassay for 411 - in renal i n s u f f i c i e n c y 411 - serum l e v e l s , influence of creatinine 411 - in serum 239,315 - urinary excretion 267 - in urine

239,315,385

D-threo-neopterin (monapterin) L-erythro-neopterin

251

Neopterin clearance

263

Neopterin l e v e l s - in a l l o g r a f t r e c i p i e n t s 263 - in autogressive diseases 263

251

1027 -

and c e l l mediated immunity 263 i n c e r v i c a l cancer 263 in diseases 263 and i n t r a c e l l u l a r pathogen i n f e c tions 263 - and other l a b o r a t o r y parameters 263 Nerve growth f a c t o r

287

Nerve terminal a u t o r e c e p t o r s Neuroblastoma c e l l

lines

Neuroblastoma N1E-115 Neurological

Phenylalanine hydroxylase 185,201,335, 363,391 - cofactors for 111 - c o n s e r v a t i o n of epitope 351 - deficiency 395 - inhibition 359 - i r o n content 363 - monoclonal antibody 359 - monoclonal a n t i b o d y , i d e n t i f i c a t i o n of epitope 351 - mechani sm 51 - pH dependence 355

347

283

283

disorders

Phenylalanine hydroxylase proteine 185

391

Neurospora c r a s s a 743,929 - p o l y g l u t a m a t e - d e f i c i e n t mutants

Phenylketonuria

243

- atypical

form

385

N e u r o t r a n s m i t t e r amine m e t a b o l i t e s - homovanillic acid 395 - HPLC 395

Phenytoin

495

-

Phosphate

5-hydroxyindoleacetic acid

Neurotransmitters

395

Nigrostriatal

215

N i t r o u s oxide

481,709,917

- i n h i b i t i o n of methionine

107

synthase

NMR, d i h y d r o f o l a t e reductase NMR, two dimensional

769®^

743

373

729

Osteosarcoma c e l l s

977

O x i d a t i v e demethylations Oxidative stress Oxygen a c t i v a t i o n

Pediococcus c e r e v i s i a e

567

495

Phenylalanine 239,315,385,395 - enzymatic h y d r o x y l a t i o n 185 - tyrosine ratio 315

865

571

by p t e r i d i n e

P h y l o g e n e t i c comparison

613

ir-Electrón d é l o c a l i s a t i o n , e f f e c t on t e t r a h y d r o p y r a z i n e r i n g conformers 85 539

a

275

969

Planarity

Peptococcus a s a c c h a r o l y t i c u s Phénobarbital

l a b e l i n g reagents

Pigmentation

283, 377

399

Phosphoribosylpyrophosphate - folate deficient 897 - in HL-60 c e l l s 897 - methotrexate-treated 897 - phosphate-induced e l e v a t i o n 897 Photoaffinity labeling 575,613

pK

51

893

P h o s p h a t e - e l i m i n a t i n g enzyme

Pig jejunum

893

547

PC12 pheochromocytoma

897

Photosensitization, derivatives 99

0 1 i g o n u c l e o t i d e - d i r e c t e d mutagenesi s 769 Ornithine

559

Photoaffinity

201, 893

Norepinephrine

pH g r a d i e n t

Phosphate, c o v a l e n t

385,523

Nickel-complex with p t e r i n s

Non-heme i r o n

92

stimulating

851

Plasma, f o l a t e c o n c e n t r a t i o n s Plasmids

733

- with encoded DHFR Platelets

Platinum c a t a l y s t 31

P-NMR

Poly G

789

475 893

655

91

495

1028 Pterin requiring enzymes, mechanism of reaction 13

Polyglutamates - a -COOH linked 737 - hepatic synthesis 485 - synthesis in vitro 929 Polyglutamation

953

- of antifolates

993

Polyglutamination

763

Polyglutamylation, regulation Poly-Y-glutamyl Pregnancy

derivatives

583 989

239,467

Prenatal diagnosis

Prodrug Promoter

309

495

733 671

NIO-propargy1-5,8-dideazafolic acid 645,993 N10-Propargyl-5,8-dideazafolic acid polyglutamates 675 Prostitutes

769

Pteridine granule

271

Pteridine pattern, effect of 3-hydroxykynurenine 323 Pteridines 339 - in amniotic fluid 239,309 - biology of 331 - biosynthesis 335 - in chronic uraemia 315 - hydrogénation 91 - side chain chemistry 31 - in thrombozytes 431 - trimethylsilyl derivatives - in urine 431 - in white cells 431 - yellow-green

Pterin-6-carboxylate Pterin deaminase

Purine, synthetic activity Pyrimidodiazepine 91

115

Pteridine symposia, history

1

151

activity 539

381

65

981

51

6-Pyruvoyl-tetrahydrobiopterin, 169,299 - deuterium exchange 299 - H-NMR 299 - mechanism of formation 299 6-Pyruvoyl-tetrahydrobiopterin 299 6-Pyruvoyl-tetrahydropterin 319

305

Pterin-dependent reactions

hydrolase

Pteroyl- y - p o l y g l u t a m a t e s - purity 65 - synthesis 65

115

Pteridines X,X1,X2,X3

743

Pteroyl polyglutamates - analysis 447 - biodégradation 743 - chain length alteration 743 - E. coli, synthesis in vitro 737 - in folate deficient rats 743 - in hepatoma eel Is 743 - interaction with enzymes 743 - intestinal absorption 743 - in sarcoma 180 cells 743

427

Proton donation

671

Pteroylhexaglutamate, cofactor in the TS cycle 763 Pteroylpolyglutamate

77

Pteroyl polyglutamate patterns - in alcohol-fed rats 447 - in liver regeneration 447 - in quail organs 447 - in rat fetuses 447 - in rat organs 447 - in starvation 447 - in Tyzzers disease 447

835

Propargyl

Pteroylglutamate coenzyme Pteroylglutamates 517 - biosynthesis from E. coli

Prenatal diagnosis, 6-pyruvoyltetrahydropterin synthase deficiency 399 Primidone

Pterins 151,251,305 - in animal liver 319 - chemistry 55,121 - GC/MS 151 - and human illness 443 - photosensitization by 99 - stereoselective hydrogénation - triplet state 99

6-Pyruvoyl-tetrahydropterin 151,169

PPH. 4

synthetase

141, 151, synthase

1029 - a c t i v i t y in - a c t i v i t y in 399 - deficiency, 399 - i n man 141 - properties

animal l i v e r 319 (fetal) erythrocytes prenatal

diagnosis

Rhodium DI0P c a t a l y s t s

91

Riboflavin, biosynthesis R i b o f l a v i n synthase Ring s t r a i n e d p t e r i n s

51

141 Saccharomyces c e r e v i s i a e

Queuine

Sarcosine

205,339

Quinespar, structure

57

SCF/ST0-3G

969

SCF/3-21G

85

Scorpion f l y

Quinoid 6 , 6 - d i s u b s t i t u t e d pterins 391

S e n i l e dementia

6,7-dimethyldihydro-

305

S e p i a p t e r i n reductase antibody

spectroscopy,

Quinonoid 6 - m e t h y l d i h y d r o p t e r i n , s y n t h e s i s and p r o p e r t i e s 81

Serine

865

S e r i n e hydroxymethyl 607

R a b b i t a n t i - F B P immunoglobulin Radioimmunoassay Raman s p e c t r a Rat l i v e r

Serotonin

639 495

763 743,

169,523 523

Side chain c h e m i s t r y , p t e r i d i n e s Silylation

31

91

S i n g l e t oxygen, p r o d u c t i o n by p t e r i d i n e derivatives 99

363

513

Stereochemistry,

Reconstitution,

synthetase

Serine hydroxymethyltransferase 887 Serum f o l a t e

729

Rat n u t r i t i o n RDA

607

819

Rat, f o l a t e c o n c e n t r a t i o n s

151

Sep-Pak, C18, r e l a t i v e a d s o r p t i o n of pteridines 107

81

Rabbit a n t i b o d i e s

815

327

S e p i a p t e r i n reductase 169,291,309 - a c t i v i t y in animal l i v e r 319 - a c t i v i t y in e r y t h r o c y t e s 399 - inhibition 169 - in man 141

p-Quinonoid-dihydropterin - conformers 85 - molecular geometries 85

isomerization

SDS Page, p u r i f i e d DHFR

Sepiapterin 125,151,271,305 - n i c k e l complex 107

Quinonoid d i h y d r o b i o p t e r i n , s y n t h e s i s and p r o p e r t i e s 81

pterin, synthesis,

271

Sepialumazine

Quinonemethides, r e d u c t i v e identification 687

893

85

Quinoid d i h y d r o p y r i m i d o d i a zepine 51 dihydro-

865

505,593

S a r c o s i n e dehydrogenase

Quinazolines 997 - synthesis, properties

Quinonoid

103,227

103

haemopoietic

419

hydride t r a n s f e r

901

Red blood c e l l s

471

S t e r e o s e l e c t i v e hydrogénation of p t e r i n s mechanism 77

Red c e l l

523

Stopped-flow f l u o r i m e t r y , DHFR

folate

Regulation

Streptococcus faecium

219

Renal f o l a t e c l e a r a n c e Retina

Retinoic acid

489

5 - s u b s t i t u t e d - 5 - d e a z a a n t i f o l ates Superoxide dismutase

279 287

Superoxide r a d i c a l

73 99

769

655,743 1001

1030 Synergism Tapeworm

(6S)-Tetrahydrobiopterin, synthesis, spectroscopy and conformational study 77

671 651

T cells - DNA s y n t h e s i s 209 - IL-2 signal transmission - pteridine synthesis TE85 c e l l s

T e t r a h y d r o f o l ate 603 - determination of 973 209

209

Ternary complex

887

Tetrahydrofolic acid - deaza d e r i v a t i v e s 953

287

- s u b s t r a t e f o r DHFR

691

- with thymidylate synthase Ternary complex a s s a y s

973 743

Tetrahydrofolate synthase

977

Teratoma 402A

- response to methotrexate T e t r a h y d r o f o l a t e reductase

639

447

Tetradecanoyl e s t e r (TPA) 431 Tetrahydrobiopterin 151,201,215, 219,291,305,319,373,391,431,835 - analogs 377 - c a t a b o l i s m 305 - in dopamine s y n t h e s i s 347 - formation in c e l l s 209 - IL-2 internalization 209 - loading test 315,385 - in man 141 - metabolic r o l e 185 - as % of t o t a l b i o p t e r i n 243 - and r e l a t e d compounds, b i o s y n thetis 125 - S phase p r o g r e s s i o n 209 - synthesis, alteration 315 - s y n t h e s i s , maturation 239 - treatment wi th 399 Tetrahydrobiopterin biosynthesi s 183,299 - i n bovine adrenal medulla 151 - hormonal r e g u l a t i o n 219 - regulation 283 Tetrahydrobiopterin deficiency 243,247,391,399 - c l i n i c a l presentation 385 - t r a n s i e n t form 239,385 - treatment 385 T e t r a h y d r o b i o p t e r i n load t e s t , h e t e r o g e n e i t y i n human response 407 (6R)-Tetrahydrobiopterin, synthesis, s p e c t r o s c o p y , X - r a y a n a l y s i s and conformational study 77 6-R-L-erythro-5,6,7,8-tetrahydrobiopterin - analogues of 111 - deficiency diseases 111

799

Tetrahydromethanopterin T e t r a h y d r o p t e r i di n - conformers 85 - molecular geometry

743

85

Tetrahydropterin regeneration

287

Tetrahydropterin(s) 151,835 - absolute c o n f i g u r a t i o n 77 - bioavailability 835 - b i o s y n t h e s i s in r a t b r a i n 169 - cation radical 73 - conformers 85 - 6,6-disubstituted 391 - e l e c t r o c h e m i c a l d e t e c t i o n of HPLC 151 - HPLC-EC 169 - i n t e r n a l redox r e a c t i o n f o r g e n e r a t i o n 151 - tyrosine hydroxy!ation

377

Tetrahydropteroylglutamate

517

Tetrahydropteroylpolyglutamates T e t r a h y d r o p y r a z i ne - conformers 85 - molecular geometry

887

85

T e t r a h y d r o p y r a z i n e - r i n g conformers, e f f e c t of i r - e l e c t r o n - d e l ocal i s a t i o n

85

Tetrahydropyrazin-ring d e r i v a t i ves, s t e r i c r e p u l s i o n , H-H 85 Tetrahydroquinoxaline

73

Thiamine, l e v e l s i n e p i l e p t i c s Thrombocytes, p t e r d i n e s i n Thymidylate, i n t r a c e l l u l a r 973 Thymidylate c y c l e - t r i t i u m r e l e a s e assay - in v i t r o 671

527

431 synthesis

671

Thymidylate synthase 631,639,645,655, 659,671,691,743,757,945

1031 -

autocatalytic s p l i c i n g 613 cooperativity of dlIMP binding 663 Ehrlich a s c i t e s carcinoma 663 expression, in vivo vs. in v i t r o 613 influence of pH 651 L 1210 663 l a c t o b a c i l l u s casei 659 mouse thymus 663 normal/tumour, comparison 663 p h o t o a f f i n i t y l a b e l l i n g 613 phyllogenetic comparison 613 role of 5-FdUMP phosphate hydroxyl 667 subunit composition 651 tapeworm, hymenolepsis diminiuta 651 temperature dependence 663 ternary complex with 639 T4 phage 613

Thymidylate synthase i n h i b i t i o n 659,663,667,675,989 - CB 3717 763 - 5-FdUMP 651 - mercaptoethanol 651 - methotrexate polyglutamates 763 - MTX analogs 985

Triazine glutamates, as a n t i f o l a t e s 959 Tri functional enzyme

901

1

6-(1',2 ,3'-Trihydroxybuty1) 257 Trimethoprim

671,769,823

Trimethylsilyl Trimetrexate

91 945

T r i p l e t state, of pteridine d e r i v a t i v e s 99 T r i s effect

115

Tritium release assay, for thymidylate cycle 671 tRNA function

205

tRNA guanine-transglycosylases tRNA transglycolase Tryptophan

339

185,391 Tryptophan hydroxylase - immunohistochemical s t a i n i n g 351 - monoclonal antibody 351 Tryptophan 5-monooxygenase - activation 369 - iron free 369

Thyroidectomy

- requirement of Fe^"1"

Thyroxine

Tumors

905

Tissue culture

547

Toluenesulfonylchloride tion 603 Torsion angles T 4 phage

activa-

969

757

Transcription Transfection

369

369

205,953

"Two-tier" hypothesis, regulation

215

T-lymphozyte subsets - circadian rhythm 411 - on physical exercise 411 a-Tocopherol

339

335

Thymidylate synthase reaction, model studies 687 219

pteri n

Tyrosine

Tyrosine hydroxylase 185,201,391 - cofactors for 111 - immunohistochemical s t a i n i n g 351 - monoclonal antibody 351 Tyrosine hydroxylation 377 - pteridine dependence 377 Tyrosine 3-monooxygenase

201,347

Ubiquinone 339 excretion Urinary folate

485, 489

733 925

Transport - lOE-lOdAm, lO-Me-lOdAm 855 - FMTX 861 - f o l i c acid 559,563,567 - methotrexate 855,861 - 5 1 -methyltetrahydrofolic acid 559

447

315,335

Urine, pteridines in 431 Valproate

495

Vinylogous-amide resonance Vitamin B i ?

481,713

85

1032 Vitamin B^2 d e f i c i e n c y Vitamin Bj2 l e v e l s i n 527

719 epileptics

Western b l o t , c r o s s - r e a c t i v i t y DHFR's 815 White c e l l s , p t e r i d i n e s in Xanthine o x i d a s e Xanthopterin

655

125,431

X-ray crystallography Yeast

865

431

799,839

of

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P r o f . P e t e r G. B e a r d s ! D e p t . of P e d i a t r i c s Yale U n i v . Sch. M e d . 3 3 3 C e d a r St. New H a v e n , C o n n . U.S.A.

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Chemistry and Biology of Pteridines Proceedings of the 5th International Symposium on Chemistry and Biology of Pteridines. Konstanz, West Germany, April 14-18,1975. Editor

W. Pfleiderer

1975.17 cm x 24 cm. XVI, 949 pages. Numerous illustrations. Hardcover. DM 190,-; approx. US $90.50 ISBN 311005928 2 The proceedings of the 5th International Symposium on "Chemistry and Biology of Pteridines" present results in pteridine research as well as some reviews of special subjects in this field. The chemical, biochemical and biological aspects of the recent investigations are discussed in detail as for example the enzymology of various important enzymes involved in the biosynthesis of pteridines in bacteria and mammalian systems, the cofactor activity of hydrogenated pteridine derivatives, the role of folate binding proteins and the transport of folate compounds into mammalian and bacterial cells. The chemistry of pteridines includes new synthetic approaches, azaanalogs, electrochemical, spectroscopical and stereochemical investigations, redox behaviour of tetrahydro forms and their rearrangements. The part of the naturally occurring pteridines consists of new components, interesting pigments, their structural elucidations, their distribution in nature as well as their potential biological activities.

Chemistry and Biology of Pteridines Pteridines and Folic Acid Derivatives Proceedings of the Seventh International Symposium on Pteridines and Folic Acid Derivatives Chemical, Biological and Clinical Aspects St. Andrews, Scotland, September 21-24,1982 Editor

J. A. Blair

1983.17 cm x 24 cm. XXXVI, 1070 pages. Numerous illustrations. Hardcover. DM 280,-; approx. US $133.30 ISBN 311008560 7 The book contains a combination of review articles prepared for the nonspecialist reader and research papers describing the most recent research work. It is regarded as a useful reference work for those whose interests lie in the fields of pteridines and folic acid biochemistry.

Prices are subject to change w i t h o u t n o t i c e

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Verlag Walter de Gruyter & Co., Genthiner Str. 13, D-1000 Berlin 30, Tel.: (030),2 6005-0, Telex 184027 Valter de Gruyter, Inc., 200Saw Mill River Road, Hawthorne, NY 10532, Tel.: (914) 747-0110, Telex 64 6677

Biochemical and Clinical Aspects of Pteridines, Volume 4 Cancer • Immunology • Metabolic Diseases Proceedings • Fourth Winter Workshop on Pteridines, February 23-March 2,1985. St. Christoph, Arlberg, Austria Editors

H. Wachter, H. Ch. Curtius, W. Pfleiderer

1985.17 cm x 24 cm. XXI, 686 pages. Numerous illustrations. Hardcover. DM 350,-; approx. US $166.70 ISBN 311010182 3 The fourth volume in this series deals with the latest developments in chemistry, biochemistry analysis, metabolism'and immunological implications of pteridines. Highlights are contributions on the electrochemistry of pteridines, on new findings regarding biosynthesis of tetrahydrobiopterin and on the close relationship between neopterin production by monocytes - macrophages and interferon-gamma. Thus, the volumes combines fundamental findings from basic sciences with new and important implications for clinical progress. Contents (Main Chapters) Chemistry and Analysis of Pteridines • Biochemistry and Metabolism of Pteridines • Tetrahydrobiopterin Deficiencies, Diagnosis and Therapy • Pteridines in Immunology • Pteridines in Cancer and other Diseases • Miscellaneous • Author Index • Subject Index. Also

available

Biochemical and Clinical Aspects of Pteridines, Vol. 1 Editors

H. Wachter, H. Ch. Curtius, W. Pfleiderer

1982. XV, 373 pages. DM 150,-; approx. US $71.50

ISBN 311008984 X

Biochemical and Clinical Aspects of Pteridines, Vol. 2 Editors

H. Ch. Curtius, W. Pfleiderer, H. Wachter

1983. XV, 435 pages. DM 190,-; approx. US $90.50 ISBN 311009813 X

Biochemical and Clinical Aspects of Pteridines, Vol. 3 Editors

W. Pfleiderer, H. Wachter, H. Ch. Curtius

1984. XII, 514 pages. DM 220,-; approx. US $104.80 ISBN 311010163 7 Prices are subject to change without notice

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\fertag Walter de Gruyter & Co., Genthiner Str. 13, D-1000 Berlin 30, Tel.: ( 0 3 0 ) , 2 6 0 0 5 0 , Telex 1 8 4 0 2 7 Walter de Gruyter, Inc., 200Saw Mill River Road, Hawthorne, N.Y. 10532, Tel.: (914) 747-0110, Telex 64 6677