Vitamin B12: Proceedings of the 3rd European Symposium on Vitamin B12 and Intrinsic Factor, University of Zurich, March 5–8, 1979, Zurich, Switzerland 9783111510828, 9783110076684

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Vitamin B 12

Vitamin B12 Proceedings of the Third European Symposium on Vitamin B12 and Intrinsic Factor. University of Zurich, March 5-8,1979 Zurich, Switzerland

Editors B. Zagalak • W. Friedrich

W DE G

Walter de Gruyter • Berlin • New York 1979

Editors

Boleslaw Zagalak, Dr. habil. sc. nat., Dr. phil. University of Zurich Zurich, Switzerland Wilhelm Friedrich, Prof. Dr.-lng. University of Hamburg Hamburg, F. R. of Germany

CIP-Kurztitelaufnahme

der Deutschen

Bibliothek

[Vitamin B] Vitamin B 1 2 : proceedings of the 3. Europ. Symposium Vitamin B 1 2 and Intrinsic Factor, Univ. of Zürich, March 5 - 8 , 1979, Zürich, Switzerland; [in memory of Robert Burns Woodward, April 10,1917-July 8,1979]/ed. by B. Zagalak; W. Friedrich. - Berlin, New York: de Gruyter, 1979. ISBN 3-11-007668-3 NE: Zagalak, Boleslaw [Hrsg.]; European Symposium on Vitamin B 1 2 and Intrinsic Factor < 0 3 , 1 9 7 9 , Zürich>; Universität < Z ü r i c h > ; Woodward, Robert Burns: Festschrift

Library of Congress

Cataloging

in Publication

Data

European Symposium on Vitamin B 1 2 and Intrinsic Factor, 3rd, University of Zürich, 1979. Vitamin B 12 . Bibliography: p. Includes index. 1. Vitamin B 1 2 -Congresses. 2. Intrinsic factor (Physiology)-Congresses. I. Zagalak, Boleslaw, 1936- II. Friedrich, Wilhelm, 1913III. Title. QP772.C9E871979 612'.399 79-25086 ISBN 3-11-007668-3

© Copyright 1979 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: Karl Gerike, Berlin. - Binding Lüderitz & Bauer, Buchgewerbe GmbH, Berlin. - Printed in Germany.

In Memory of Robert Burns Woodward April 10,1917-July 8,1979

Preface

The papers in this volume appear in their original format as presented at the Third European Symposium on Vitamin B 1 2 .The Symposium, which was organized by B. Zagalak and presided over by Lord Todd, took place at the University of Zurich, Switzerland on March 5 - 8 , 1 9 7 9 . In the tradition established in Hamburg by the First and Second European Symposia, the Third Symposium brought together scientists involved in the many different aspects of Vitamin B 1 2 chemistry and physiology. The Symposium was attended by 350 participants from 25 countries. We are indebted to D. Arigoni, J. Dunitz, A. Eschenmoser and V. Prelog fortheirguidance. We also wish to thank J. Borschberg, Marion Cavazzi, J. Penton, C. von Schlabrendorff, Birgit Senkpiel and Maria-J. Zagalak for their invaluable assistance in the preparation of this volume.

July 15,1979

B. Zagalak and W. Friedrich

VIII

o=c

COENZYME B

C O N T E N T S

Chapter 1.

CHEMISTRY OF CORRINOIDS. TOTAL SYNTHESIS OF VITAMIN b

Lord Todd, O.M., P.R.S. - Introductory ropean Symposium on vitamin B^

remarks

Crowfoot

analysis R.B. Woodward

-

A. Eschenmoser

Synthetic - Chemical

and recent R.V. Stevens

Hodgkin

of vitamin

of 3rd

Eu1

K. Folkers - Introductory remarks. Historical isolation of crystalline vitamin B^ Dorothy

for opening perspectives

on the 7

- New and old problems

in the

structure

B^

19 vitamin

synthesis

B^^

37

of corrinoids:

current

problems

advances

- Recent

studies

H.H. B^^ Inhoffen - Alew reactions derivatives

89 on the synthesis

of vitamin

of the chromophoric

B. Grlining and A. Gossauer - Structure called stable yellow corrinoids

system

and reactivity

B^^ .... of

of the

G. Schlingmann and V.B. Koppenhagen - Concerning the structure called yellow products obtained from naturally occurring free corrinoids

vitamin

119 137

so141 of sometal149

G. Schlingmann, B. Dresow, L. Ernst and V.B. Koppenhagen - Correlation of vitamin B^ methanolysis products with cobyrinic methyl ester amides prepared from hydrogenobyrinic acid amides ....

155

B. Dresow, V.B. Koppenhagen and W.S. Sheldrick - Preparation and X-ray structural analyses of a cobyrinic acid diamide and the corresponding rhodium analogue

159

X

W. Friedrich - Concerning

a new corrinoid

from municipal

sludge

M. Fountoulakis, J. Retey, W.E. Hull and B. Zagalak - steric as studied by 1H-NMR of the substitution by vitamin B troscopy

. .

163

course spec169

P. Engel, G. Rytz, L. Walder, U. Vogeli and R. Scheffold - Synthesis and X-ray analysis of a porphyrin-type Co(I)-complex . . . . . .

171

G. Rytz, L. Walder and R. Scheffold - Stereochemistry of the formation and cleavage of the Co-C bond in a vitamin B^ model ...

173

E. Jorin, A. Schweiger and Hs.H. GLinthard - The structure oxocobalamin, a single crystal ESR study

175

of

super-

R.K. Gupta, P.C. Goswami and A. Nath - NMR studies of a novel of vitamin B I.P. Rudakova, T.A. Pospelova, E.M. Tachkova and A.M. Yurkevich Circular dichroism study of organocobalamins

form 179 183

M. Moskophidis - Concerning electronic, CD and ORD spectra of isomeric pairs of several (Co-methyl)-corrinoids

189

V. Popova and S. Angel ova - Some properties ferrate

193

of

cobalaminhexacyano-

K.L. Brown, A.W. Awtrey, P.B. Chock and S.G. Rhee - Temperaturejump kinetics of the "base-on" - "base-off" equilibrium of methylcobalamin J.M. Saveant, N. de Tacconi, D. Lexa and J. Zickler - Electrochemistry of vitamin B^^. Equilibria, kinetics and mechanisms in the B,„ - B,_ - B,_ oxido-reduction 12a 12r 12s D. Lexa, J.M. Saveant and J.P. Soufflet - Electrochemistry of vitamin B^g. Alkyl cobalamins and cobinamides

Chapter 2.

199

203

213

BIOSYNTHESIS OF CORRINOIDS

A.R. Battersby - Recent biosynthetic

researches

A.I. Scott - Intermediary

of cobyrinic

metabolism

on vitamin B^

...

217

acid biosynthesis.

247

G. MLiller, R. Deeg, K.D. Gneuss, G. Gunzer and H.-P. Kriemler - On the methylation process in cobyrinic acid biosynthesis

279

V.Ya. Bykhovsky - Biogenesis of tetrapyrrole and corrinoids), and its regulation

293

compounds

(porphyrins

M. Imfeld, D. Arigoni, R. Deeg and G. MLiller - Factor I ex dium tetanomorphum: proof of structure and relationship tamin B7 biosynthesis

Clostrito vi315

XI

P. Renz, J. Horig and R. Wurm - On the biosynthesis methylbenzimidazole moiety of vitamin B^

of the

317

J. Horig and P. Renz - The enzyme system of propionic transforming H.C. Friedmann

riboflavin

5,6-di-

acid

bacteria

into 5,6-dimethylbenzimidazole

- Straight approaches

to the nucleotide

W. Walerych and E. Pezacka - Ribosomal biosynthesis

proteins

323

loop

....

share in vitamin

E. Pezacka and W. Walerych - The ribosomal proteins L2S involved in vitamin B^ biosynthesis

345

L2, L5, L18 and 359

K. Sato, S. Inukai, S. Ueda, T. Seki and S. Shimizu - Formation and role of vitamin B^ in Protaminobacter ruber and Rhizobium meliloti

Chapter 3.

331

361

CORRINOID DEPENDENT ENZYMES AND REACTIONS. MODEL SYSTEMS

R.H. Abeles - Current status of the mechanism coenzyme D. Arigon i - A stereochemical action

approach

of action of

B^. . .

to the diol dehydratase

373

re389

S. Fukui and T. Toraya - Coenzyme B^^-dependent diol dehydratase distribution and metabolic role %n Enterobacteriaceae, enzymological properties and interaction with coenzyme B^

413

A.A. Poznanskaya and T.L. Korsova glycerol dehydratase interaction logs

431

B. Zagalak - Glycerol dehydratase

Adenosylcobalamin-dependent with substrates and their ana-

from Aerobacter

aerogenes

....

J. R6tey - The mechanism of action of methylmalonyl-CoA mutase studied with isotope labelling and synthetic models B.M. Babior - Recent studies on the mechanism amine ammonia-lyase

of action of

437

as 439

ethanol461

M.R. Hollaway, H.A. White, K.N. Job!in, A.W. Johnson, M.F. Lappert and O.C. Wall is - Studies on the mechanism of reactions catalysed by ethanolamine ammonia-lyase

471

P. Diziol, H. Haas, J. R6tey, S. Graves and B.M. Babior - Steric course of the ethanolamine-ammonia lyase reaction

485

H.P.C. Hogenkamp - The chemical synthesis and coenzymatic of analogs of adenosylcobalamin in the ribonucleotide reaction

489

properties reductase

XII

J.R. Pilbrow - Review of the EPR of B 7 9 reactvons

and B

IGJP

•

-dependent

enzyme

-¡-¿J

r n

r

bUb

G.N. Schrauzer, J.H. Grate, M. Hashimoto and A. Maihub - Vitamin s awe-vent problems and recent advances

; 511

H.G. Wood - The role of corrinoids in the total synthesis of acetate from C02

529

J.M. Wood and Y.-T. Fanchiang - Mechanisms for B^^-dependent ation

539

P. Dowd - Nonenzymic models for the enigmatic coenzyme carbon-skeleton rearrangements

methyl-

B^^-dependent

D. Dolphin, A.R. Banks, W.R. Cullen, A.R. Cutler and R.B. The mechanism of action of coenzyme B^

557 Silverman575

M.P. Atkins, B.T. Golding and P.J. Sellars - Model systems for adenosylcobalamin

dependent enzymic reactions

587

J.A. Robinson - Bridged cobaloximes as vitamin B^

models

599

D.L. Anton and H.P.C. Hogenkamp - Modified adenosylaobalamin: model systems for the active sites of corrinoid-dependent enzymes . .

Chapter

4.

A N A L O G U E S AND A N T A G O N I S T S OF VITAMIN

605

B12

D. Perlman, K.L. Perlman, T.H. Williams, U. Schbmer and Y. Izumi Naturally occurring vitamin B^ antagonists and their potential therapeutic value

609

T. Kamikubo and M. Hayashi - Structures of some vitamin Banalogues and their biological as well as biochemical functions . .

625

V.B. Koppenhagen, E. Warmuth, G. Schlingmann and B. Dresow - Novel metal-free corrinoids and metal analogues

635

R. Bieganowski and W. Friedrich - Preparation Fe(III¡-analogue of vitamin B^

647

Chapter

5.

VITAMIN

B

of ferribalamin,

the

ASSAY

B.A. Cooper, J. Peyman, Erika Jonas and V.M. Whitehead - vitamin B^2 assay: an evaluation of radiodilution assay using cobinamide to increase specificity

649

J.A. Begley and C.A. Hall - Effect of residual extract products and the type of binders (R or IF) on serum vitamin B^ levels by radioisotope dilution assay

655

XIII

N.A. Sourial and D.L. Moll in - Differential assay of aobamides in serum using R-protein radioisotopic dilution assay, E. aoli and E. gracilis assays

657

D. Jacobsen, R. Green and M. Savage - Rapid determination rinoids by high performance liquid chromatography

663

P. Gimsing,

E. Hippe and Ebba Nex0 - Determination

balamins B. Zagalak

by one-dimensional - Chemical

thin-layer

quantitation

of

cor-

of the plasma

co-

chromatography

665

of corrinoids

671

Z. Schneider - Enzymatic estimation of vitamin B^ R.A. Beck - Essential prerequisites for the analysis of cyanocobalamin in biochemically complex samples using radiometric competitive binding assays

Chapter 6.

675

ABSORPTION, TRANSPORT AND DISTRIBUTION OF VITAMIN B

D.M. Matthews - Distribution J.H. Fendler - Vitamin considerations C. Bradbeer

673

B^

2

of cobalamins in membrane

and practical

- Transport

in the animal

mimetic

agents

-

body

...

theoretical

applications

of vitamin

B^

695

in Escherichia

C.A. Hall - The plasma transport of cobalamin (Cbl) R. Grasbeck - Soluble and membrane-bound vitamin B^ teins

coli

. . . . .

vitamin

B^-binding

J.P. Nicolas - Heterogeneity pernicious anaemia Y. Parmentier, traluminal

proteins

711 725

transport

pro743

B. Rachmilewitz, M. Schlesinger, R. Rabinowitz and M. Rachmilewitz The origin and clinical implications of vitamin B^ binders the transcobalamins R. Carmel - Large man serum

681

and complexes

in

765

hu777

of antibodies

against

B^

binders

G. Marcoullis, J.P. Nicolas and M.O. Perrin phase of vitamin B^ transport in humans

in 791

- The

in803

Marijke Frater-Schroder, R. Osusky, W.H. Hitzig, R. Butler and Aila Hasler-Hakkinen - Polymorphic variants of transcobalamin II rare alleles in family studies

807

W. Fenrych, J. Hansz, K. Boduch and B. Pytlak - Relationship between cobalophilin releasing and functional state of polymorphonuclear granulocytes

813

XIV

M. Inada, M. Kameyama and M. Toyoshima - The significance min B^g binders in the central nervous system

of vita821

H. Gilbert - In vitro acid dissociation of cobalamin-transcobalamin II complexes formed in vivo: a probe in the study of TC Il-kinetics

827

C.M. Becker and W.S. Beck - Calcium dependencies in the binding of transcobalamins to subcellular particles of liver cells ....

833

C.H. Tan and T.J. Broekelmann - The effect of antibiotics cobalamin II synthesis in rabbit liver cell culture

837

on trans-

Ebba Nex0, M.D. Hollenberg and H. Olesen - Solubilization and characterization of the transcobalamin II acceptor from human placenta and rabbit liver

843

Ebba Nex0, H. Olesen and Marianne R. Hansen - Strength of binding of methyl-, 5 '-deoxyadenosyl-, cyano- and hydroxocobalamin to human transcobalamin I and II and intrinsic factor

851

J. Lindemans, J. van Kapel and J. Abels - Adsorptive endocytosis of transcobalamin II-vitamin B^ by isolated rat liver parenchymal cells

855

Mary Haus, Pamela D. Green and C.A. Hall - Species specificity tween TC II, TC II-Cbl uptake and anti-TC II

861

be-

H.J. Porck, Marijke Fräter-Schröder and A.K. Häkkinen - Transcobalamin II polymorphism in african populations

863

R. Green, P. Myers and D. Jacobsen - Transcobalamins during induction of nutritional cobalamin deficiency in the fruit bat . . .

867

U.-H. Stenman and E.-M. Salonen - Heterogeneity of transcobalamin demonstrated by isoelectric focusing in urea

873

II

Marijke Fräter-Schröder, P. Vitins and W.H. Hitzig - Radioimmunosorbent determinations of unsaturated and total transcobalamin II in human serum

877

K.C. Das and V. Herbert - The "dU suppression test" and "thymidine suppression test": evidence for reciprocal relationship between the "de novo" and "salvage" pathways of DNA synthesis

881

J.F. Burman, P.N. Malleson, N.A. Sourial and D.L. Mollin - TC II deficiency: observations with deoxyuridine suppression test . . .

889

Ebba Nex0, H. Olesen and Marianne R. Hansen - Spectral studies on hog intrinsic factor and hog non-intrinsic factor

895

D. Bucher, J. Thomsen, Ebba Nex0 and H. Olesen - Amino terminal quence of hog non-intrinsic factor and hog intrinsic factor

se. .

905

I. Kouvonen and R. Gräsbeck - Subunit structure of the pig ileal intrinsic factor receptor

909

XV

J.A. Begley, A. Trachtenberg and C.A. Hall - Cobinamide assay for intrinsic factor M. Inada, M. Toyoshima and M. Kameyama vitamin B^ in enterocytes

blocking 917

- Intracellular

transport

of 919

P.C. Sharpe and M.A. Horton - Evidence for the chief cell as the source of intrinsic factor secretion in the rat

921

E. Hauptmann - Long-term treatment with intrinsic factor

929

of pernicious

anemia

patients

R. Gräsbeck, I. Kouvonen, Margaretha Lundberg, R. Tenhunen and Hannele Yki-Järvinen - An analogy of cobalamin membrane transport: an intestinal receptor for heme

933

E. Hippe, P. Gimsing and N.H. Holländer - A simplified method quantitative determination of vitamin B^ absorption

939

for

R. Morishita and H. Uchino - Vitamin absorption studies with gastroenterological plastic whole body counter in patients with operation

945

D. Jacobsen and Y. Montejano - Affinity of cobalamin-binding proteins

949

photo-release

M. Katz and E. stopa - Effect of pentagastrin tion in the guinea pig

purification

in vitamin B^

absorp955

W.G.E. Cooksley, J.A. Owens, J.C. Linnell, H.A. Hussein, J.M. England and A.S. Tavill - Synthesis of cobalamin coenzymes in the rat and their secretion from the liver into plasma and bile

961

Ebba Nex0 and P. Gimsing - The pattern rocytes

967

of cobalamins

in human

J.A. Begley and C.A. Hall - Presence of sulfitocobalamin tracts. Resolution and identification by SP-Sephadex exchange chromatography

eryth-

in cell exC-25 cation 971

W.S. Beck, R. Cohen and J. Jorgensen - Mitochondrial cobalamins: types, sources, and functions with evidence of their noninvolvement in mitochondrial DNA synthesis

Chapter

7.

PHYSIOLOGY

AND

PATHOLOGY

OF

VITAMIN

K. Folkers, J.Y. Choe, Y.Z. Kwen, S. Shizukuishi Studies on vitamin B^ and ubiquinone J.M. England and J.C. Linnell - Haematological deficiency

975

B]2

and S. Nishii -

aspects

979 of

cobalamin 991

XVI

E.H. Reynolds

- The neurology

of vitamin B^

deficiency

1001

W.H. Hitzig, Marijke Fräter-Schröder and R.Seger - Clinioal diseases related to defioienoies of vitamin B^ transport proteins

1009

M. Inada, M. Ogawa, H. Kameyama and F. Udaka - Effects of methylaoexperimentally-induced balamin on peripheral neuropathies or neuropathies

1017

R. Baumgartner and H. Wick - Inherited deficiencies of the deoxyadenosylcobalamin (Ado-Cbl) dependent L-methylmalonyl-CoA mutase system: clinical and biochemical aspects

1019

Sarah Hopper - An investigation in ribonucleotide reduction

1025

of a possible role for coenzyme in rabbit bone marrow

B^

A. Stroinski - Participation of cAMP in regulation of coenzyme dependent glycerol dehydratase synthesis from Klebsiella pneumoniae ATCC 85955

1029

G.B. Diekert, E.G. Graf and R.K. Thauer - Carbon monoxide oxidation by Clostridia : evidence for the involvement of a corrinoidlike compound

1033

J.A.L. Amess, J.F. Burman, M.H. Cullen and D.L. Mollin - The in vivo effects of nitrous oxide on human bone marrow - a morphological, biochemical and cell cycle study

1037

E.V. Quadros, Beverley Jackson, A.V. Hoffbrand and J.C. Linnell Interconversion of cobalamins in human lymphocytes in vitro and the influence of nitrous oxide on synthesis of cobalamin coenzymes

1045

Rosemary Deacon, M. Lumb, M.Muir, Janet Perry, I. Chanarin, Minty, M.J. Halsey and J.F. Nunn - Studies on cobalamin late metabolism in rats exposed to nitrous oxide (N^0)

1055

Barbara and fo-

B. Reed, J. Dinn, S. McCann, P. Wilson, H. O'Sullivan, D.G. Weir and J.M. Scott - Alterations in mammalian cells induced by inactivation of vitamin B^ with nitrous oxide

1061

Shirley W. Thenen - High ascorbic in the rat

1065

acid intake and vitamin B^

status

V. Herbert, L. Landau, R. Bash, S. Grosberg and N. Colman - Ability of megadoses of vitamin C to destroy vitamin B^ and cobinamide j?aand to reduce absorption of vitamin B^ (with a note on B dioassays) J. Skupin, K. Waliszewski and B. Jaszewski - Interaction between methylcobalamin and some amino acids in single cell proteins

1069 .

1079

S.J. Kanopkaite, J.A. Rackus, V.R. Kalnev and V.A. Stankevicius Methylcobalamin and the modification of proteins in vitro . . .

1085

L.E. Taraseviciene and S.J. Kanopkaite - Deoxyadenosylcobalamin the process of methylation of tRNA in model systems

1091

and

XVII

E.P. Jordan, N.V. Antoshkina and L.I. Vorobjeva - The participation of coenzyme B^ in the synthesis of DNA by Propionibacterium shermanii

1095

J.C. Linne!1 and D.M. Matthews - Recent advances in cobalamin metabolism: abnormalities in coenzyme distribution in tumor development and in inherited metabolic disease

1101

G.R. Brazenas, V.V. Bartkeviciene, A.M. Aleksiene, A.J. Alioniene, S.J. Kanopkaite and V.F. Zovyte - On some properties of the effect and metabolism of different cobalamins in tumor-bearing rats

1113

L. Elsborg and J. Mosbech - Gastric cancer as a risk factor in pernicious anaemia

1119

N.V. Myasishcheva, E.V. Quadros, Y.V. Vares and J.C. Linnell - Interference with cobalamin metabolism and tumour growth by an analogue of methylcobalamin

1125

K. Fujii, T. Nagasaki and F.M. Huennekens - Vitamin B1S requirement for replication of mouse leukemia L1210 cells: functional relevance to folate metabolism

1127

F.I. Haurani - Vitamin B^

1131

and folia acid interrelations:

a new look

E.L.R. Stokstad, Margaret May-Sheng Chan, J.E. Watson, T. Brody and L. Jaenicke - The effect of hypothyroidism on histidine oxidation and folate-dependent enzyme levels

1139

H. Sauer - Cobalamin-dependent methionine synthesis and the regulation of the metabolism of activated folic acid derivatives in cultured human lymphoblasts

1145

Author and Citation

1149

Index

Subject Index Collection of Photographs

1171 from the Symposium

1213

INTRODUCTORY REMARKS FOR OPENING OF 3rd EUROPEAN

SYMPOSIUM ON VITAMIN B ] 2

by Lord Todd, O.M., P.R.S.

Just about a month ago at the Royal Society in London I listened to a fascinating lecture on the coelacanth - that remarkable primitive fish believed to have been extinct for several million years but which came up from the depths of the Indian ocean on a deep water fisherman's line a few years ago.

What the lecturer did not tell us, of course, was what the

coelacanth felt when it was hauled up from the deep and exposed to public view.

Perhaps I could give some indication of that now, since today,

greatly honoured as I am to preside over the Third European Symposium on Vitamin B ^ »

I feel a little bit like a coelacanth - a kind of living

fossil dredged up from the past days of Vitamin B ^ »

securely hooked by

our efficient secretary, Dr. Zagalak, and put up for public examination. It is, for me, difficult to believe that thirty-one years have passed since Vitamin B ^

was isolated in pure form and that my connection with

the search for the anti-pernicious anaemia factor goes back some ten years before that.

Arising from my work on Vitamin B^ in the mid-thirties

I naturally developed an interest in the Vitamin B group and in the connection between some at least of its members (at the time unisolated)

© 1979 Walter de Gruyter & Co., Berlin • New York Vitamin Bis

2

and the apparently complex and certainly confusing picture presented by the various forms of anaemia.

But my first contact with the problem of

pernicious anaemia itself was in 1938.

Early in that year I visited the

United States (for the first time incidentally) and stayed for a few days with an old friend of my wife's family, Dr. H. D. Dakin (Dr. Folkers at least will remember him!) at his home on the Hudson River near Ossining where he had a private laboratory in the grounds of his large house.

Dakin, a well-known biochemist, had been endeavouring to track down the anti-pernicious anaemia factor, or factors in liver extracts ever since the discovery by Minot and Murphy (1926) that whole liver therapy was effective in controlling the disease.

I well remember those few days

during which he told me at length about his work and sought to encourage me to enter the field.

Despite all his efforts Dakin (like other workers

in the field) had made little headway and his progress was very slow indeed.

For this I think the main reason was the great difficulty of

biological assay.

Extracts could only be tested on human patients suf-

fering from pernicious anaemia.

Clear cut clinical cases of the disease

were not very common and the results of tests - as in all cases where small numbers of human subjects are used - were unreliable.

In addition,

of course, clinicians were, not unnaturally, more interested in curing their patients than in testing fractions of extracts.

I recall Dakin

being particularly cross with one clinician who gave a patient the test material and then - just to make sure - gave a good dose of whole liver as well!

That may have been an extreme case, but certain it is that when

a material on test was administered, there was in some clinics a tendency to expect immediate results; if these were not evident then the known specific - whole liver - was administered without further delay and no test result was obtained.

These difficulties, of course, beset all work-

ers in the field.

At this time I was acting as scientific adviser to Glaxo Laboratories Ltd., then a smallish company pushing its way from baby-foods into the vitamin

3 business, and after I got back from America I took an increasing interest in its work on whole liver extracts.

In charge of the fractionation of

the extracts was Dr. Lester Smith who, almost single-handed, kept plugging away at the pernicious anaemia problem.

I was, of course, in contact with

although not actively engaged in the pernicious anaemia research although I did some work on nutritional anaemia factors in the B complex where I confess I was narrowly anticipated in the identification of folic acid and pantothenic acid.

I do not complain about that however because the

B complex work took rather a back seat during the war; any time I had to spare from war work in those years was devoted mainly to the nucleotide field.

It was, I suppose, in 1946 that the possibility that the factor being sought in liver might be coloured was first seriously discussed by us.

At

any rate I recall a meeting at the Glaxo laboratories where Lester Smith mentioned that his best fractions from liver concentrates seemed to have a pink colour.

In the absence of any test other than the clinical one it

was decided to set about isolating the coloured material checking it from time to time in the clinic.

The idea proved sound and in 1948 Smith

and Parker were able to report the isolation of crystalline Vitamin B ^ . They had in fact been narrowly anticipated by Folkers and his co-workers at Merck & Co., Rahway who had been able to use a microbiological assay for the vitamin using Lactobacillus lactis Dorner and who also found that the vitamin could be obtained much more easily from a fermentation using Streptomyces griseus.

It was also about the same time that Jukes and his

colleagues at Lederle Laboratories in their pursuit of the so-called "animal protein factor" also independently isolated the vitamin from the fermentation broth of Streptomyces aureofaciens.

When the vitamin was isolated at Glaxo Laboratories it was decided that I should study it chemically at Cambridge while Dorothy Hodgkin would go ahead with X-ray crystal!ographic studies in Oxford, the two groups remaining in contact with each other and with Lester Smith.

It wasn't easy

u to do chemical work at that time if only because the tiny amount of material we had in the early days came from liver and only slowly did we get material from the much more prolific fermentation source. the problem of material

In addition to

supply there was also the complication that

turned out to be a substance of frightening complexity.

B^

I doubt whether

more than a handful of you in this audience can imagine what it was like to tackle a substance like B ^

chemically without the help of n.m.r. mass

spectrometry or even modern infrared spectrometry.

This is not the place

to recount all the trials and tribulations that ensued.

I was lucky in

my colleagues and especially in Prof. A. W. Johnson who with me conducted the Cambridge work and we did indeed furnish key products for X-ray analysis which helped Dorothy Hodgkin to bring the structural work to a brilliantly successful

conclusion.

Even then chemical

over for, as we all know, the cobamide

surprises were not

coenzymes were discovered con-

taining covalent cobalt-carbon bonds, a thing hitherto undreamed of. Thereafter, although Professor Johnson has continued work in the B-^ field I effectively ceased research on it apart from a series of studies on pyrroline-N-oxides and their reactions begun with a view to their possible use in Vitamin B ^

synthesis; this work too was dropped in the mid-

sixties in the light of the more promising and indeed ultimately

success-

ful synthetic studies of Woodward and Eschenmoser.

Since the establishment of structure in 1955 a prodigious amount of work has been done on Vitamin B ^ chemical and chemical.

and related products - nutritional, bio-

Nutritionally and biochemically the interest and

value of such work is evident and through it our knowledge of enzyme processes and of the manifold activities of corrinoids are steadily unfolding.

But what of the chemical work?

With most of the other vitamins the

major contribution of chemistry was to render them accessible.

This is

not to say that no contributions were made to chemistry as such but the main result was the increase in vitamin accessibility. not been the case in the B ^

This has certainly

group for the brilliant synthetic studies of

Woodward and Eschenmoser have certainly not provided a cheap route to B 1 ? .

5 But the great mass of work it entailed has enormously enriched our armoury of synthetic methods - an

enrichment which has had and will continue to

have a profound effect in the whole field of synthetic chemicals for all sorts of purposes. B^

Moreover, one should not forget that it was work on the

synthesis that provided the inspiration which gave us the Woodward-

Hoffmann rules on the conservation of orbital symmetry - surely the most important advance in organic chemical theory since the concept of dynamic stereochemistry was introduced.

Not surprisingly the unusual structural features observed in the Vitamin B-|2 molecule have stimulated studies on the biosynthesis of the corrinoids and we will be hearing about some of the striking findings in that field during our symposium.

One cannot help wondering whether investigations

on biosynthesis may throw light not just on the way in which the B ^

mole-

cule and those of the related corrinoids are put together, but why these molecules have been selected to perform their function in nature.

Is

there something intrinsically valuable - something unique about them or are they to be regarded as accidents of evolution?

These and many other puzzles will be discussed at length during our symposium and it is my hope that these discussions may lead to still further advances in our knowledge of structure and function in the Vitamin group.

B^

INTRODUCTORY REMARKS

HISTORICAL PERSPECTIVES ON THE ISOLATION OF CRYSTALLINE VITAMIN

K.

Folkers

I n s t i t u t e f o r Biomedical Research, A u s t i n , Texas 78712

The U n i v e r s i t y of Texas a t

I t was 3 1 y e a r s a g o , on December 11, 1947, f r o m a q u e o u s a c e t o n e a s a r e s u l t of team a t Merck, Edward R i c k e s , Wood. 8

t h a t v i t a m i n B^j

Austin,

crystallized

t h e combined e f f o r t s o f my r e s e a r c h

Norman B r i n k , F r a n k K o n i u s z y , and Thomas

My t o t a l e f f o r t , which was i n t e r r u p t e d by t h e w a r ,

spanned

about

years.

With t h e a p p r o v a l o f P r o f e s s o r Z a g a l a k f o r t h e s e i n t r o d u c t o r y r e m a r k s , s h a l l r e c a l l some h i g h l i g h t s of t h e i s o l a t i o n s of v i t a m i n B ^

which may

h a v e some i n t e r e s t ,

even importance,

r e s e a r c h of

T h i s i s o l a t i o n may seem s i m p l e t o d a y , b u t o v e r 30

y e a r s ago, high

today.

and p e r h a p s a p p l i c a b i l i t y

t h e i s o l a t i o n was f o r m i d a b l e , c o m p l e x ,

I

for

controversial,

the

and of

risk.

I t appeared

t o me i n t h e e a r l y

'40's

that

p e r n i c i o u s anemia f a c t o r was i m p o r t a n t .

t h e g o a l of i s o l a t i n g t h e I n my p r e s e n t a t i o n ,

a t a Con-

f e r e n c e on "The C h e m i s t r y and P h y s i o l o g y of G r o w t h " which was i n t i o n of t h e B i - C e n t e n n i a l o f P r i n c e t o n U n i v e r s i t y "Now t h a t related

i n 1946 ( l ) ,

t h e c h e m i s t r y of p t e r o y l g l u t a m i c a c i d and

c o n j u g a t e s i s known,

it

is likely

a n t i p e r n i c i o u s anemia f a c t o r ( s ) .

The unknown f a c t o r

f a c t o r s f o r p e r n i c i o u s anemia i n l i v e r and o t h e r today,

if

i m p o r t a n c e i s j u d g e d by t h e amount of usefulness."

© 1979 Walter de Gruyter & Co., Berlin • New York Vitamin Bit

liver or

sources

are possibly the outstanding u n i d e n t i f i e d f a c t o r ( s ) e v i d e n c e on t h e r a p e u t i c

its

t h a t more

r a p i d p r o g r e s s w i l l be made on t h e i s o l a t i o n of t h e

of

clinical

anti-

celebra-

I said:

a Little did I know how very important this factor would ultimately be to growth.

Vitamin B 1 2 had been said to have caused a near-revolution in the

commercial raising of poultry and livestock. In discussing my initial proposed research with Dr. Randolph T. Major, who was my boss, to work on the isolation of the antipernicious anemia factor, he was unimpressed, and reminded me that many others had not succeeded over a long period of time.

Some thought the extrinsic factor was multi-

ple, and there was some conflict of the relative importance of the intrinsic and extrinsic factors} both of these aspects discouraged effort.

Dr.

Major said that he and Dr. Hans Molitor had considered the project, but had decided against it. Director.

However, Dr. Major was a flexible and considerate

He said he would discuss my desire with Dr. Henry Dakin, who

was a member of the Merck Board of Directors.

It was not unusual in those

years for research decisions to be made at the Board level, and there were members on the Board who were qualified to make research decisions.

Some-

times, I wonder today if there should not be more scientists on Boards of Directors.

Dr. Dakin was an extraordinary person, and we all regarded him

with great affection.

He, like Dr. Major, was considerate and

his

response was something like, — " o f course, let Karl have a go at it."

—

and — " i shall send him a bottle of one of my initial liver fractions." He had worked on this problem years before. I had learned from Dr. Major the wisdom of attending medical meetings in addition to meetings of the American Chemical Society, and so I went to a medical meeting in Atlantic City. in the lobby of the Claridge Hotel. about my research.

Fortuitously, I met Randolph West, M.D., We knew each other, and he asked me

I said that I wanted to work on the isolation of the

antipernicious anemia factor, and to try chromatography to achieve further purification.

He asked, "what is chromatography?"

He offered no judgment

on the technique, but promptly said he would test my fractions.

Then,

he impressed me about the hardship I could experience, because there would be so few patients with pernicious anemia available even in the metropolitan area of New York.

Dr. West was on the faculty in Hematology at

the College of Physicians and Surgeons of Columbia University.

I was also

learning to cooperate with physicians, which I have done during all of my

9

career. On my r e t u r n t o t h e l a b , teered

I reported

t o D r . M a j o r t h a t D r . West had

t o t e s t my c h r o m a t o g r a p h i c f r a c t i o n s f r o m a c r u d e l i v e r

D r . Major was s a t i s f i e d ,

and

approved.

F r a n k K o n i u s z y and I d i s s o l v e d a l i t t l e an a q u e o u s medium, and p a s s e d

of t h e b l a c k i s h l i v e r r e s i d u e

on i s o l a t i n g a l k a l o i d s by a l u m i n a c h r o m a t o g r a p h y .

Alu-

experience

The e l u a t e f r o m t h e

b e c a u s e t h e b l a c k " j u n k " of t h e l i v e r r e s i d u e had

s t u c k t o t h e t o p of t h e a l u m i n a c o l u m n . anemia f a c t o r m i g h t n o t be s t a b l e , p e r i e n c e , we l y o p h i l i z e d

Fearing that the

antipernicious

and f o r t h e f i r s t t i m e i n o u r l a b .

something — the e l u a t e from the alumina

The r e s i d u e was " s n o w - w h i t e " , t e s t on t h e f i r s t a v a i l a b l e

to

What h a p p e n e d when D r . West l o o k e d

a t t h e " s n o w - w h i t e " s a m p l e was a b o u t a s f o l l o w s :

He i m m e d i a t e l y Folkers has

— "Fantastic,

t h e a n t i p e r n i c i o u s anemia f a c t o r " .

called

already

Such a p r o n o u n c e m e n t

c a u s e d D r . Dakin t o t e l e p h o n e i m m e d i a t e l y t o D r . M a j o r f o r a r e p o r t , an e x p l a n a t i o n on why D r . M a j o r h a d n ' t r e p o r t e d s u c h an i m p o r t a n t to him.

Dr. Major r e p l i e d

would g e t a r e p o r t f r o m me. and a s k e d

ex-

column.

and an a l i q u o t was m a i l e d t o Dr. West

patient.

D r . Dakin and s a i d s o m e t h i n g l i k e : crystallized

in

t h e s o l u t i o n o v e r a column of a l u m i n a .

mina was a v a i l a b l e a t Merck, and F r a n k and I had had l i m i t e d column was c o l o r l e s s ,

volun-

residue.

and

event

t h a t he knew n o t h i n g a b o u t i t , b u t t h a t he Within minutes,

I was c a l l e d

to h i s

office

t o e x p l a i n why I h a d n ' t i n f o r m e d him - my b o s s - t h a t I had

crystallized

the f a c t o r , but I d e t e c t e d a "twinkle

s p o n s e was s o m e t h i n g l i k e :

in h i s eye".

— " i h a v e n ' t done any s u c h t h i n g J "

My r e Then,

remembered h a v i n g s e e n t h e w h i t e i c e p a t t e r n of t h e l y o p h i l i z e d and e x p l a i n e d

t h a t t h i s a p p e a r a n c e of a c r y s t a l l i n e

r e m i s s i o n of t h e anemia i n a p a t i e n t . unknown t h e n i n t h e Merck l a b s . ,

indeed,

residue,

s t r u c t u r e must have

b e e n m i s t a k e n l y viewed by D r . West a s t h e " c r y s t a l l i n e D r . West t e s t e d s u c h a r e m a r k a b l e s a m p l e a n d ,

I

factor".

Obviously, it

found t h a t

C h r o m a t o g r a p h y was

caused

essentially

and I m i g h t h a v e b e e n t h e f i r s t

chemist

t h e r e t o u s e t h e t e c h n i q u e i n such r e s e a r c h .

The i n t e l l e c t u a l

t h e s e e v e n t s was t r u l y momentous.

t h a t t h e a c t i v e f a c t o r had

I realized

i m p a c t of

p a s s e d t h r o u g h a column of a l u m i n a , which i t was n o t e x p e c t e d

t o do on t h e

b a s i s of a p r o t e i n - l i k e

in the

c o m p o s i t i o n which was c l e a r l y e v i d e n t

liter-

1D

ature.

So, the factor appeared not to be a peptide or protein, and it

appeared certain that the factor could be purified by chromatography. Randolph West immediately wanted more fractions to test.

Dr. Dakin and

Dr. Major became more enthusiastic, and my resolve to pursue the isolation to the goal was established. succeed.

I never really worried about how we would

To work was the resolve.

I went to Chicago and ordered crude liver extracts equivalent to tons of liver.

The Accounting Department of Merck considered cancelling my order,

which was extraordinarily expensive in their experience, but Dr. Major again supported me.

Dr. West established a clinical network in all of

New York City to find pernicious anemia patients for tests. Over time, we learned that chromatography by various procedures was highly successful, that the factor was relatively stable, that it could be distributed between certain solvents, that it was of relatively low molecular weight, and that it was not a protein.

These were some of the identified

basic properties which guided our purification, and recognition of such properties were vital to our continuing success. I had always been acutely aware of the need to discern a chemical or physical property which could correlate with activity and circumvent the need for clinical tests, except for confirmation of activity. literature of past examples for strategies. a strategy.

I studied the

We continuously speculated on

Eventually, we had many positive fractions from Dr. West's

clinical tests, and a few inactive ones, because he was not receptive to testing fractions which were likely to be inactive.

He was justified in

not wanting to administer a fraction projected to be inactive to a patient in relapse.

We did make progress, and were not too far from the end,

although we didn't know it. One day, I took a trip to the University of Maryland to visit Dr. George Briggs, and to discuss cooperation on pantothenic acid. about the regulations of his University.

I asked George

Dr. Briggs took a rejected pro-

posal from his desk which he said would illustrate the contractual conditions of the University for cooperation with an industry.

The proposal

had been written by Dr. Mary Shorb, and was based on very limited micro-

11

b i o l o g i c a l data w i t h L . l a c t i s Dorner and crude l i v e r e x t r a c t s .

She had

sought f i n a n c i a l aid from two prominent Pharmaceutical Companies that a c t u a l l y had programs of r e s e a r c h on the a n t i p e r n i c i o u s

anemia f a c t o r , and

from one Foundation t h a t funded research on n u t r i t i o n .

Her data were so

limited,

and i t was so i n c r e d i b l e — so v e r y u n b e l i e v a b l e ,

that t h e r e could be a r e l a t i o n s h i p between the growth of the h e m a t o l o g i c a l response of

— at t h a t

time,

an organism and

a p a t i e n t w i t h p e r n i c i o u s anemia, t h a t

two i n v o l v e d companies and the foundation d e c l i n e d t o support h e r .

the It

is

i n t e r e s t i n g today t o e v a l u a t e the " s e c r e t " o f the f a i l u r e s and successes of

such c r u c i a l

decisions.

I e x p l a i n e d t o George B r i g g s and Mary Shorb that l a t i o n of t h e a n t i p e r n i c i o u s

anemia f a c t o r ,

I was working on the

iso-

and could p r o v i d e her w i t h

both a c t i v e and i n a c t i v e l i v e r f r a c t i o n s from c l i n i c a l t e s t s which would o r would not i n d i c a t e a p o s s i b l e c o r r e l a t i o n o f t h e two a c t i v i t i e s . e s s e n t i a l l y f o r g o t about pantothenic my v i s i t

acid which had been the b a s i s

I for

t o Dr. B r i g g s .

I r e a l i z e d on my r e t u r n t r i p that those at Merck who acted on o u t s i d e g r a n t s could a l s o be n e g a t i v e .

I saw Dr. M a j o r e a r l y the next morning,

recounted my d i s c u s s i o n w i t h Drs. B r i g g s and Shorb, and asked f o r h i s approval of

a sum so small t h a t he might immediately say " y e s " t o my

d e s i r e t o f i n a n c e assays by Mary Shorb on our f r a c t i o n s f o r only a few months.

I explained that

I could r e a d i l y t e l l

if

t h e r e were a promising

c o r r e l a t i o n o r not between t h e growth o f her microorganism sponse o f t h e anemia t o our f r a c t i o n s . the great

s i g n i f i c a n c e of t h e p o t e n t i a l c o r r e l a t i o n .

mindedness and u n d e r s t a n d i n g , he r e p l i e d , about 400 d o l l a r s .

and t h e

Both he and I r e a d i l y

"Yes".

re-

visualized

True t o h i s

open-

The sum might have been

There was no committee a c t i o n — j u s t two chemists

no medical a d v i c e — j u s t

a l a b . man and h i s b o s s .

One may c o n t r a s t

s i t u a t i o n w i t h t h e decision-making of management and of committees,

this today.

Dr. S h o r b ' s assay was v e r y promising and d i d e x p e d i t e the f i n a l steps isolation. more of

—

of

However, her assay was then so h i g h l y q u a l i t a t i v e t h a t i t was

a t e s t than an a s s a y .

The assay p a r t i c u l a r l y made p o s s i b l e the

i n i t i a t i o n of the search f o r t h e f a c t o r i n f e r m e n t a t i o n m a t e r i a l s

as a

12

practical source for commercialization.

I had anticipated such a source,

but could never have justified giving Dr. West a fraction from fermentation to inject into a human being.

Although INDs from the Food and Drug Admin-

istration were not in existence then, we were as cautious then as we are today even with an IND.

My personnel on the project was increased, on the

basis of the apparent microbiological assay, and the need to include fermentation materials in the research. The isolation culminated on December 11, 1947, when Ed Rickes noticed beautiful red crystals forming in a blood-red solution which was derived from a fermentation broth. (Photograph 1)

PHOTOGRAPH 1 FRANK KONIUSZY

KARL FOLKERS

EDWARD RICKES

NORMAN BRINK

THOMAS WOOD

13

P r e v i o u s l y , we had become s i g n i f i c a n t l y the red c o l o r and a c t i v i t y .

aware of the r e l a t i o n s h i p between

The c r y s t a l l i n e v i t a m i n was achieved both

from the f e r m e n t a t i o n source and from l i v e r only a couple of days or so apart.

There were no experimental r e s t r i c t i o n s

the f e r m e n t a t i o n f r a c t i o n s , but the h i g h e s t

of

of economy on working w i t h l a b o r a t o r y economy was

necessary f o r t h e manipulation of the b e s t l i v e r f r a c t i o n s .

The e x c e p -

t i o n a l judgment and e x p e r i m e n t a l s k i l l of Dr. Norman B r i n k , who obtained the c r y s t a l s of vitamin B I 2

from the l i v e r f r a c t i o n s , more than matched

t h e i r paucity and high f i n a n c i a l The f i r s t

clinical test

f o r everyone.

value.

of the c r y s t a l l i n e v i t a m i n was indeed a h i g h l i g h t

P r i o r i t y was g i v e n t o t e s t i n g f i r s t c r y s t a l s from

p r i m a r i l y f o r medical reasons, but t o some e x t e n t f o r i n t e r i m

liver,

industrial

s e c u r i t y on the c r y s t a l s from f e r m e n t a t i o n . Norm Brink made a s o l u t i o n of t h r e e micrograms of the c r y s t a l s f o r t i o n by Dr. West t o a p a t i e n t ,

and he l y o p h i l i z e d the a l i q u o t .

injec-

When he

showed me the ampoule I could see n o t h i n g , because t h r e e micrograms of

the

substance was i n v i s i b l e u n t i l one i n s p e c t e d the ampoule with a m a g n i f y i n g lens.

I doubted t h a t Dr. West would f a v o r a b l y respond t o an "empty ampoule"

and I asked Norm t o put some p h y s i o l o g i c a l so that Dr. West could at l e a s t

s a l i n e s o l u t i o n in the ampoule

see a s o l u t i o n .

A l s o , we showed Dr. West

data t o j u s t i f y our recommendation that he administer a dose as small as t h r e e micrograms t o a p a t i e n t .

With Dr. West, we reminisced and c o n t r a s t e d

that f i r s t w h i t e r e s i d u e t o the new red c r y s t a l s . Rahway, and Dr. West searched f o r a p a t i e n t .

Norm and I went back

my home and d e s c r i b e d the strong and prompt hematopoetic response. succeeded. one p a t i e n t .

to

In due t i m e , he c a l l e d me at

No one e v e r doubted the c l i n i c a l v a l i d i t y

We had

of the response of

Today, one might have f i r s t t o do s a f e t y tests on rodents and

even a double b l i n d t r i a l

at t h e c l i n i c a l

l e v e l — even f o r hematology.

Dr. West died soon a f t e r he t e s t e d the c r y s t a l s , but not b e f o r e he was honored by h i s medical p r o f e s s i o n f o r h i s important

role.

S e v e r a l months b e f o r e t h e day the vitamin c r y s t a l l i z e d , here in Z u r i c h , s i t t i n g m a i l from the l a b .

I had been

right

i n the garden of the Baur au Lac H o t e l reading my

Dr. M a j o r had sent me on a t r i p t o Europe.

Norm Brink

had w r i t t e n to me that medical a d v i c e had been v e r y u n f a v o r a b l e f o r con-

Ik tinuing the research on the isolation of the factor.

I thought I knew the

identity of the physician behind the negative pressure, and it was influential.

Also, commercial people in the company had not been sympathetic

to my research, because it was not apparent how the company could make a profit from treating a rare disease.

My faith and that of Dr. Major in

the potential profit value to the company of a new vitamin kept us willful. X firmly believed that Dr. Major would not stop my project while X was in Europe at his request, and he did not do so. On my return, he said something like —"Just take it easy". might have said "Keep a low profile".

Today, he

In only a few more months, the red

crystals were visible in a centrifuge tube, witnessed by all, including George Merck and other executives. In retrospect, I am confident that we would have succeeded to isolate the crystalline vitamin without the advent of L. lactis Dorner, because we were so conscious of the strategy to correlate some property with activity. This property became the red color which was not only important to us, but to all others who subsequently isolated the red vitamin.

Dr. Shorb's assay

was important and ultimately became quantitative} one executive, examining our data, said that we isolated the substance not because of the assay but in spite of it.

I tell students today not to worry about their bad assays,

but to anticipate that the assay will become good after they have completed their isolation, and no longer need the assay! I wrote the essential parts of three companion manuscripts for publication in Science.

One was by Rickes, Brink, Koniuszy, Wood, and myself, on cry-

stalline vitamin B 1 2 j one was on the microbiological data by Dr. Mary Shorb, and the third was on the clinical data by Dr. West.

Before sub-

mission of the manuscripts for publication to Science, Dr. Major asked me if a pre-publication copy of our manuscript on crystalline vitamin BI 2 could be sent to Glaxo in England.

We knew that revealing by a pre-publi-

cation copy of the information that the antipernicious anemia factor had been crystallized and that it was red in color would obviate the need for clinical tests on fractions for Glaxo, except for confirmation of clinical activity.

All that was then necessary was to look for a red band on a

15

chromatographic column and isolate it.

This vitamin very readily crystal-

lizes . The submission of the manuscripts to Science was doubtless facilitated by a fortuitous meeting which X had with Dr. Fritz Lipmann in Atlantic City during a scientific meeting.

He walked to me on the boardwalk and asked if

we had indeed isolated a new vitamin for pernicious anemia.

The "news" had

started to "leak" out of Columbia university, and witholding publication any longer was about like trying to hide a big building on fire.

The three

papers appeared in the issue of April 16, 1948, of Science. To designate the red crystalline substance — factor, —

the antipernicious anemia

was unsatisfactory, because the expression is cumbersome.

sought a new name.

Names were concocted and discarded.

I

I preferred the

name vitamin B with the next unused number in vitamin nomenclature as a subscript.

A check with Dr. Crane of Chemical Abstracts revealed no pub-

lication or unpublished abstract recording vitamin BI 2 .

Neither Dr. Crane

nor I could have known that Professor Hauge of Purdue University had a manuscript in press with the expression vitamin B 1 2 .

Professor Hauge told

me later that when he saw our paper in Science, he had to change all the twos to threes in the galley of his paper, which subsequently appeared with the expression "vitamin BI 3 ." used the designation vitamin BI 3 .

I often wondered whether I would have For Professor Hauge, this designation

was ill-fated. Per Laland of the Nyegaard Company of Oslo was an inspired and dedicated researcher who had previously sought the antipernicious anemia factor. (Photograph 2)

He fractionated liver extracts and his fractions were

tested by Jens Dedichen, M.D., on patients.

The German invasion of Norway

terminated Per's research and almost ended his life. continue his fractionation, he might have succeeded. was a man of great enthusiasm and kindness.

Had Per been able to Per died recently.

He

On the basis of friendship

between his company and Glaxo in England, I understand that Lester Smith of Glaxo was provided with the Norwegian data from Per's research which had been terminated by the invasion.

Then, Lester Smith and his associates

made their own pioneer contributions to the purification from liver.

16

PHOTOGRAPH 2 PER LALAND

KARL FOLKERS

Lester Smith and L.F.J. Parker presented an account of their purification of the antipernicious anemia factor, and their obtaining of a crystalline product as red needles from aqueous acetone at a meeting of the Biochemical Society at Oxford on May 29, 1948 (2).

In their abstract they stated that

— " i t appears probable that our substance is the same as that recently described by Rickes, Brink, Koniuszy, Wood, and Folkers (1948) with the suggestion that it be given the name vitamin B 1 2 " . Smith and Parker not only achieved crystalline vitamin B12, second crystalline entity which was the hydroxycobalamin. much credit.

(Photograph 3)

but also a Lester deserves

He made important observations, and he was

also initially restricted by clinical tests on patients performed by

17

PHOTOGRAPH 3

LESTER SMITH

Dr. C.C. U n g l e y . The c r y s t a l l i z a t i o n s investigators

of vitamin B 1 2 by Smith and Parker and l a t e r by o t h e r

in the UK and elsewhere in Europe and in t h e USA were aided

by knowing that the v i t a m i n i s r e d .

I t s ease of c r y s t a l l i z a t i o n

from

18

aqueous acetone was an asset t o e v e r y o n e . We at Merck a l s o made p i o n e e r i n g c o n t r i b u t i o n s t o the s t r u c t u r e o f B I 2 as d e s c r i b e d in 28 p u b l i c a t i o n s during 1948-1956. o r g a n i c chemistry of the b e n z i m i d a z o l e , phosphate of the r i b o s i d e .

We e l u c i d a t e d

and i t s r i b o s i d e ,

We c o r r e c t l y

vitamin

i d e n t i f i e d and s y n t h e s i z e d

l - a m i n o - 2 - p r o p a n o l in t h e c o r r e c t o p t i c a l l y

a c t i v e form.

the

as w e l l as the the

We a l s o had t h e

good f o r t u n e t o c l a r i f y s t r u c t u r a l l y t h a t part of t h e c o r r i n nucleus which was t h e o n l y part that was i n i t i a l l y

ambiguous in the remarkable X-ray

crystallography of Dorothy Crowfoot and her

associates.

I t was here in Zurich at a Symposium, when A l e x Todd t o l d me t h a t

the

s t r u c t u r e o f v i t a m i n B 1 2 had been f i n a l i z e d by Dorothy C r o w f o o t , and I r e c a l l h i s saying t h a t

i t might have taken " f o r e v e r " i f

t o be s o l v e d by c l a s s i c a l o r g a n i c

t h e s t r u c t u r e had

chemistry.

B e f o r e the c r y s t a l s , we n e v e r even could have dreamed about the f o r t h coming b i o c h e m i s t r y , o r about t h e B i 2 - c o e n z y m e , o r t h a t Woodward and Eschenmoser would s y n t h e s i z e t h e v i t a m i n .

However, we were a l e r t ,

at t h e

r i g h t t i m e , t o t h e r o l e of vitamin B 1 2 as the animal p r o t e i n f a c t o r , which became of g r e a t s c i e n t i f i c

and commercial

importance.

May I conclude t h i s r e m i n i s c i n g with the hope t h a t the lessons and t h e t r u t h s l e a r n e d i n t h i s e x t r a o r d i n a r y s e r i e s of e v e n t s could

expedite

current r e s e a r c h on o t h e r d i s e a s e s , because almost nothing of b a s i c

sig-

n i f i c a n c e i s known about so many d i s e a s e s .

References

1.

Parpart, Arthur K . , e d . : Chemistry and P h y s i o l o g y of Growth, ton U n i v e r s i t y P r e s s , P r i n c e t o n , New J e r s e y , 81, 1949.

2.

Smith, L e s t e r : 1969.

Prince-

Vitamin B 1 2 , Methuen and Co. L t d . , New York, 27,

NEW AND OLD PROBLEMS IN THE STRUCTURE ANALYSIS OF VITAMIN B Dorothy Crowfoot Hodgkin Chemical Crystallography Laboratory, Oxford I t was with great diffidence but also considerable happiness that I accepted the i n v i t a t i o n to attend t h i s symposium - diffidence because I have been working rather far away from B ^ problems for many years, happiness because both past and present B ^ studies are f u l l of remarkable i n t e r e s t . Dr. F o l k e r ' s description of the i s o l a t i o n of red c r y s t a l s of vitamin B 1 2 brings immediately to my mind the day in late May 1948, that Dr. Lester Smith f i r s t called in at our laboratory in Oxford.

He had himself j u s t

c r y s t a l l i s e d h i s own preparation of p u r i f i e d anti pernicious anaemia factor and he had a small sample of c r y s t a l s with him.

Since the Merck

l e t t e r to Science had recorded the refractive indices of the vitamin c r y s t a l s , he planned to take h i s specimen to Dr. R. C. S p i l l e r in the Dept. of Geology and a l s o to Dr. Mary Porter who was an expert in chemical i d e n t i f i c a t i o n from the external

forms of c r y s t a l s .

I could not r e s i s t

looking at them with Dr. Lester Smith under the microscope - they were very small but well defined needles showing marked pleochroism and I suggested attempting to take X-ray photographs of one of them s t r a i g h t away. day.

I took one photograph overnight and the second during the following Dr. Lester Smith described h i s preparation at the Biochemical

Society meeting that morning, giving a suggested molecular weight of the vitamin of about 3,000. photograph:

In the afternoon we developed the second X-ray

from the two together and a guessed crystal density we could

estimate the molecular weight as about 1,500.

In the weeks that followed, Dr. Lester Smith sent more material from which I was able to grow much better c r y s t a l s ; Merck Laboratories for direct comparison.

© 1979 Walter de Gruyter & Co., Berlin • New York Vitamin B12

samples were also sent from the One exciting day Dr. McCrea

2D from Glaxo rang me up to say cobalt was present in the vitamin, one atom for the X-ray c r y s t a l l o g r a p h i c molecular weight. heavy atom for you;

He added " T h e r e ' s a

you could determine the structure of the c r y s t a l s " .

Very s h o r t l y afterwards I was at Columbia U n i v e r s i t y and had the very great pleasure of meeting a c c i d e n t a l l y Dr. Randolph West himself and hearing of h i s experiences making c l i n i c a l

t e s t s of the Merck preparations

including other cobalt compounds. I grew larger c r y s t a l s of vitamin B ^ from water and soon r e a l i s e d that they d i f f e r e d from the a i r dried c r y s t a l s ;

the unit c e l l was a l i t t l e

larger and contained a few more water molecules; clearer and gave more d i f f r a c t i o n e f f e c t s .

the c r y s t a l s were

The dry c r y s t a l s , formed by

l o s s of water, as they were picked out of t h e i r mother l i q u o r , were always a l i t t l e cracked.

Dr. Porter, who measured them in the optical

goniometer,

described the r e f l e c t i3o n s from the faces, i l l u s t r a t e d in Figure 1, as blurred and d i s t o r t e d .

Figure 1.

A i r dried c r y s t a l s of cyanocobalamin, measured by Dr. M. W. Porter.

The detailed X-ray a n a l y s i s , carried out over the following y e a r s , establ i s h e s c l e a r l y the r e l a t i o n s h i p between the wet and dry c r y s t a l s . Figure 2a cyanocobalamin molecules are seen projected along c» c o r r i n ring system i s nearly on edge;

In

the

the amide groups project into

21 layers of water, some molecules of which, marked as f i l l e d c i r c l e s , are hydrogen bonded to the B ^ molecules, while others, open c i r c l e s ,

fill

disordered pools.

In the a i r dry c r y s t a l s , there are s t i l l water layers but the e x i t of some water molecules from the structure i s associated a l s o with amide group movements.

As the second projection, along a, shows (Figure 3) a l l

the

acetamide groups turn outwards from the c o r r i n ring in the wet c r y s t a l s . In the a i r dried s t r u c t u r e , one has swung round the bond C7-37 to contact the cyanide group and t h i s has led to other small adjustments.

Figure 2.

Projection of the c r y s t a l structure along c of (a) wet c r y s t a l s and (b) a i r dried c r y s t a l s of cyanocobalamin

22

Figure 3.

Projection of the crystal structure along a of (a) wet crystals and (b) air dried crystals of cyanocobalamin

Today, in this Symposium, we are celebrating the complete synthesis of Vitamin B ^ and I cannot resist commenting on Professor R. B. Woodward's colour photographs comparing crystals of natural and synthetic vitamin

B^.

Both crystals have the characteristic face development drawn by Dr. Porter; both also show small cracks and distortions caused by removal from their mother liquor, the rotation of the amide groups and loss of water, associated with air drying.

The characteristics of the vitamin B ^ crystals, wet and dry, are important in relation to the accurate definition of the molecular structure. In both crystals there is some disorder, associated with the mobility of the water molecules in the wet crystals and the process of removal of water from the dry crystals.

Long after we had finished the structure

analyses of the crystals, as we thought, and published them, Dr. Lester Smith gave us some marvellous large crystals he had made, obtained from acetone-water mixtures.

Dr. David Dale took two X-ray photographs of

them, (kept wet) just for the record, and found that the reflections extended much further than did those from the earlier crystals - to the limit of copper Ka radiation.

They also showed occasional very small differences

23

in the relative intensities of the X-ray reflexions compared with those earlier observed, due probably to very small differences in the equilibrium positions of the cyanocobalamin molecules in the crystals in the presence of acetone compared with those in water alone.

If someone would

measure these crystals with a modern diffractometer and refine the crystal structure with modern computing, it should be possible very greatly to improve the accuracy of bond length determination within the cobalamin nucleus over that obtained from the measurements made between 1948-54.

In the first Symposium on vitamin B ^ at Hamburg in 1956 I gave an outline of the course of the X-ray analyses of four crystals that had led us to the writing of the complete chemical structure of cyanocobalamin.

Strict-

ly, those analyses were not sufficiently accurate to provide direct evidence of the hydrogen atom positions in the molecule except in a rather approximate, imperfect way;

necessarily general chemical evidence had to

be invoked for the complete formulation presented.

In the meantime the

positions of almost all the hydrogen atoms of the cyanocobalamin structure have been observed directly through the neutron diffraction analysis of a related molecule - the acid obtained as the first product of mild acid hydrolysis of cyanocobalamin.

This acid has been variously called E2

by Dr. Lester Smith and CMS^ by Bernhauer et al. and has been isolated from sewage sludge;

It is a natural product

it may be the last stage in the

biosynthetic production of cyanocobalamin.

f

Figure 4.

Crystals of monocarboxylic acid derived from cyanocobalamin, drawn by F. H. Moore.

2U It crystallises in great slabs, 4 mm across, seen in Figure 4, very different from those of cyanocobalamin itself.

The appearance of all the atoms

in these crystals as obtained from neutron diffraction by Dr. Frank Moore, 4 Dr. Brian O'Conner and Dr. B. T. M. Willis

is shown in Figures 5a and b.

The hydrogen atoms appear as negative peaks, all the other atoms as positive peaks, nitrogen

heavy ,and cobalt

very weak, as required by the

relative neutron scattering factors.

Figure 5.

Monocarboxylic acid. Summary of the neutron scattering density synthesis (a) for the corrin nucleus and substituents projected on the mean plane of the nucleus (b) for the propanolamine, cyanide and "nucleoside" projected on the mean plane of the dimethylbenzimidazole group. The contour interval is 2.5f/A3.

Here we are in a quandary.

We first looked at crystals of the monocarbox-

yl ic acid in order to assist in finding which of the amide groups of cyanocobalamin was the first to be hydrolysed.

Lester Smith provided us

with an ethyl amide derivative to study by X-ray analysis.

However, the

crystals of this were fragile, easily decomposed and poorly diffracting; we were tempted to analyse instead the beautifully crystalline acid itself, thinking that by neutron diffraction

we ought to be able to distinguish

one acid among the amide groups of the molecule.

25 o At first all seemed well.

The first data set, which extended to 1.3A,

showed most of the atoms of the molecule just as expected; mide

all the aceta-

groups were particularly well defined, and, although the propionic

chains were much less clear, one was markedly weaker than the others, that at position e, while the remainder had small peaks - rather too many in fact - round the terminal atoms, suggesting hydrogen atoms of amide 5, 6 groups (tt - COOH-CM. CH; CH, fHvCH;-CO Sll, C (a\ NH.-CO-CH- CH .c/ (,^\ll-OliCH;COSK: KH.COCH.rC^i^iC^«. ^OCtlfCM--"i 1 a(;CHfCO NH; (el v r i«. I CH(CH Ac-Vl, ciifrS^di

Since e was the position favoured by Bernhauer, it seemed likely the problem was settled.

However, extension of the neutron diffraction data

to 1.oX and further refinement have produced a different picture. is clear indication of disorder at e:

There

two alternative positions for the

chain can be traced which account for its weakness (Figure 6).

Figure 6.

Summary of the neutron scattering synthesis for the e chain; all atoms beyond C49 are omitted from the structure factor calculation.

26 Each chain seems to terminate in one strong and one

weak peak but there

is little evidence of hydrogen atoms in negative peaks.

At the terminal

atoms of the chains b and d, some of the small negative peaks that appeared earlier

have disappeared, leaving d clearly an amide group but b with

only one hydrogen peak, an apparently well defined acid. 7 and 8).

(Compare Figures

We have to admit that now there is a discrepancy not only with

our old conclusions but also with still further chemical experiments carried out by P. Rapp, G. Bolzer and E. Fridrich, which still favour position e 7 . We also must admit that our diffraction data may still not be quite accurate enough to settle the structure for sure;

we should per-

haps once again look for a derivative of the acid.

There are a number of other crystals which we looked at in early stages of our examination and which may be worth further study.

Figure 7. Neutron scattering synthesis over a chain terminal atoms; left all atoms included; right, terminal atoms omitted in phasing calculations.

Figure 8. Neutron scattering synthesis over b chain terminal atoms; left, all atoms included; right, terminal atoms omitted in phase calculations.

One such is the selenocyanide of cobalamin.

This derivative was made for

us, as Lord Todd reminded me recently, by Dr. Stafford in his laboratory, to help us in the X-ray analysis of B ^ -

It was in the first confused

electron density maps we calculated for this compound - phased on cobalt and selenium - that we first recognised and began to believe in the characteristic feature of the corrin ring, that two five-membered rings are o directly linked together . In addition, the cobalt atom was found to be directly attached to sulphur in the thiocyanide and to selenium in the selenocyanide crystals, which is very unusual, see Figure 9.

27

Figure 9. Electron density contours over "nucleoside", cobalt and selenocyanide groups from the X-ray analys i s of selenocyanocobalamin.

Figure 10. in neo

The atomic positions

Both crystals are very marvellously pleochroic, deep green to colourless, compared with crimson to colourless in cyanocobalamin.

In the n i t r i t o

derivative, as far as I can remember, the pleochroism i s blue-violet to colourless.

One crystal which we early photographed and of which the

structure was never,as far as I know, settled, was called i s o - B ^ — formed by a i r oxidation.

Another was neo B.,,later studied by R. A. Bonnett and 9 others, and found to be the epimer of cyanocobalamin at C13 . The crystal structure of neovitamin B ^ was solved by Eric Edmond, Helen Evans and John Hodder.

I t astonished us by being very similar to that of B ^

s e l f - wet and dry.

it-

I t appears that the propionamide group can turn

either directly up or down from the corrin ring system, following d i f f e r ent puckering of the C r i n g , with almost no change in overall molecular packing, the alternative epimeric positions of the side chain being occupied by solvent. These observations bring me to the most c r i t i c a l structure analysis we carried out, that of the "red fragment" or hexacarboxylic acid, remark-

2fl able crystals of which were obtained by Cannon, Johnson & Todd in the autumn of 1953.

We first heard news of these crystals through Dr. Janet

Vaughan who had learnt of their existence from Lord Todd at a meeting they both attended.

Jenny Pickworth and I went over to Cambridge and

searched through the whole preparation Jack Cannon had obtained - the product of the alkaline hydrolysis of the vitamin, acidified and left in a mixture of solvents to crystallise during a brief holiday.

We took

about a dozen crystalline fragments from his material back to Oxford and found that one of them,

drawn in Figure 11, gave very good X-ray photo-

graphs.

J§ -

O

Î

Lf
n20\fctt/V23F/mol/S0Min H c/ _ HjC' b)-Q3t Vblt/1.12 F/mol/SO Min H'

PI/O.1 N CE/uac^ in CHjCN/AcjO/TFA (160: 20:1)

unter Argon / 0°

Ausb. «0% nebst (9% Edukt NO. 7

Figure 7: What we originally had in mind when we started the search for non-photochemical versions of the A/D-secocorrin •+• corrincycloisomerization was to provide chemical assistance to the work which had been taken up in various laboratories on the problem of vitamin B ^ biosynthesis. This search produced a whole series of new (A

D)-cyclizations; these are - without

discussion of details - summarized on this and the following Figures.

96

The first case in the series was Bernhard Kräutler's

7)

ex-

perimental solution to the problem of simulating the act of photochemical excitation by a succession of electrochemical one-electron oxidation and reduction steps. The electrochemical (A

D)-cyclization shown in the Figure is struc-

turally identical and mechanistically quite closely related 8) to the photochemical cycloisomerization

C O R R I N S BY R E D U C T I V E C Y C L I Z A T I O N

CHj CHj

OF

D-DEHYDRO-A/D-SECOCORRINATES

-0 4Volt/01nCE/Hg CHsCN/CFjCOOH U M/LiClQ, RT / Argon

Ei/j (0.tn CE / Hg /01n LiClCV RT) in CHjCN

-120Volt (reversible)

in CHjCN/ -0 3£Volt |revers'^e 10 4% CFjCOOH lirreversible 0 5»*/,«

50% + 20% dimer A. Pfaltz

B. Kräutler

No. 8

Figure 8: By adhering to the concept that any reaction sequence that would produce the elusive 1,19-dehydro-l,19-secocorrin ttsystem mentioned earlier would in fact constitute an A/Dsecocorrin

corrin-cyclization it was possible to foresee 18 that 1-methylidene-A -dehydro-1,19-secocorrinates would cyclize to corrinates by a succession of protonation and electrochemical reductive steps. This turned out to 9)be true under the reaction conditions given in the Figure

97

D-PYRROLO-CORRINS ACID CATALYZED

BY

(A-D)-CYCLOISOMERIZATION

traces of acid at RT

No. 9

B Kraoher, VRasMi.A Ptau

A.W. JOHNSON'S

(A-D)-CYCUZATIONS TO

CORROLES AND TETRAOEHYDRO-CORRINS

(1964- )

e-g.

Figures 9 and 10: In the course of the electrochemical studies on A/D-secocorrinoids a 19-acetoxy-l,19-secocorrin derivative had become available. Base-induced elimination of acetic acid therefrom led to a D-pyrrolo-l-methylidene-dehydro-1,19-secocorrinate which cyclized to the corresponding D-pyrrolo-dehydrocorrinate with extreme ease in the presence of the slightest traces of acid

. This type of A/D-cyclization corresponds

to one of A.I. Scott's earlier proposals

of how the corrin

structure might be formed in B ^ biosynthesis. In connection with a synthesis of isobacteriochlorins (see below) we have

98

recently encountered a similar cyclization in the C/D-pyrrometheno-bisdehydro series. If one extrapolates this type of cyclization to even higher states of dehydrogenation one arrives at A.W. Johnson's (A -+• D)-cyclization of dipyrromethenes to corroles and tetra-dehydro-corrinates. Figure 10 recalls a representative example of those pioneering studies of the Johnson school

12)

CORRINS BV DECARBOXYLATIVE (A—•)-CYCLIZATION

CN

No. 11

Figure 11: An (A -»• D)-cyclization that turned out to have a special impact on our further work was the decarboxylative (A -*• D)cyclization of l-methyl-19-carboxy-l,19-secocorrinates. This was originally planned as a test of whether the departure of CC>2 from position 19 of a secocorrinoid 19-carboxylic acid could provide a path to the 1,19-dehydro-l,19-secocorrin intermediate and therefore lead to a corrin. It was found that there is in fact such a decarboxylative cyclization pathway. However, it is not induced by decarboxylation but occurs by deprotonation in position 19, followed by decarboxylation after cyclization

14)^ What followed from this were the

experiments summarized in Figure 12.

99

CORRINS BY CYCLOISOMERIZATION OF 19- FORMYl -A/D - SECOCORRINATES H,c

HiC Hi OHC CH, 'OH

CN A Pfaltz

NO. 12

Figure 12: A nickel(II)-l-methylidene-19-formyl-l,19-secocorrinate was shown to cyclize under acid/base catalysis with great ease to the corresponding 19-formyl-corrinate which can be quan13)14) titatively deformylated by hydroxide ions The cyclization step is clearly the acid/base-catalyzed analog of the photochemical A/D-secocorrin •+ corrin-cycloisomerization. The originally unexpected ease with which 19-formyl-corrinates deformylate illustrates a remarkable and surprising stability of corrinoid 19-ylide intermediates.

Figure 13: The Summa of the presently available experience on the chemical synthesis of corrins clearly shows that the direct junction between the ring A and D, the characteristic structural element of corrin ligands, is not the bottleneck in the formation of the corrin structure as we had assumed at the outset of the investigation on the chemical synthesis of vitamin B,_. 12

100

Johnson's oxidative and electrocyclic (A -+• D)-cyclizations (arrow on the upper left of the Figure) to dehydrocorrinates and our photochemical A/D-secocorrin -*• corrin-cycloisomerization (arrow vis-à-vis) are but two extremes of an apparently rich spectrum of (A

D)-cyclizations and show that the

corrin ligand skeleton is inherently a broadly accessible structure rather than a synthetically narrow and demanding one. As one of the consequences of such a change in attitude towards the corrin structure we recently went so far as to ask the following radical question: Could the corrin structure have had a pre-enzymic origin

? Once we had turned our

attention in this direction, questions such as whether a newly uncovered version of (A ->• D)-cyclization might have a chance of being biomimetic were no longer our main concern and our directions in planning experiments changed accordingly. Among the new questions which we ask are the following:

101

Figure 14: What is the chemistry of hexahydroporphinoids under strict exclusion of oxygen? Which is the thermodynamically favored position of unsaturation among isomeric hexahydroporphinoids? How does this position depend upon the presence and the nature of metal ions? Does a skeletal A/D-contraction of a hexahydroporphinoid metal complex having a partially corrinoid chromophore to a true corrin complex amount to a thermodynamic sink? Are there accessible pathways for such a rearrangement?

pyrroloid

corrinoid

N o . 15

1Q2 Figure 15: Collective experience on the relative stability of corrin and corphin complexes relative to the corresponding metal-free ligands justifies the following working hypothesis: Thermodynamic equilibria between hexahydroporphinoids having pyrroloid type of chromophore unsaturation and hexahydroporphinoids having corrinoid type of chromophore unsaturation ought to lie on the side of the former in the absence, but on the side of the latter in the presence of transition metal ions. This expectation has found its first support in the results of the following experiments.

CHLORINS (t=c-2-1)

- 30% CoCt? CHjCOOH / EtjN i t Xylene /liS'/6 d

COBALT

NO OXYGEN

-30%

Ch. A n g s t

ISOBACTERIO CHLORINS (6 diastereomers)

No. 16

Figures 16 to 19: Under conditions given in Figure 16, octaethylporphyrinogen is converted into a mixture of cobalt(II) complexes which after acidolytic removal of cobalt(II) ions delivers in about equal amounts octaethylchlorin (trans/cis mixture, ratio 2 to 1) and a mixture of diastereomeric isobacteriochlorins in which the trans-trans-trans isomer predominates. After chro-

103

'H-NMRratios

ttc tcc

ctc ccc

ISOBACTERIOCHIORIN MIXTURE

c o l u m n chromatography H P L C separation crystallization

No. 18

matographic separation the binary mixture of the trans-transtrans and the trans-cis-trans diastereomer can be obtained in crystalline form. The stereochemical assignment of these two main isomers is based on the ~*"H-NMR spectrum of corresponding mixtures of their bispyrrolic dihydroderivatives obtained by

101+

No. 19

hydrogenation in the presence of platinum. The protons of the methylene group between the pyrrolic rings C and D are homotopic in the trans-trans-trans isomer and give rise to a singlet, whereas the corresponding protons of the trans-cistrans isomer are diastereotopic and give rise to an AB-system (see Figure 18). Air oxidation of the mixture gave pure transoctaethylchlorin, showing as expected that we are not dealing with the pair of cis-trans-cis and cis-cis-cis isomers. The crystalline material has been identified by spectral comparison with the "isobacteriochlorin" obtained by a procedure described originally by Eisner

and later by Inhoffen

,

namely by reduction of iron(II)-octaethylporphinate or -chlorinate with sodium in isopentyl alcohol. Eisner's "isobacteriochlorin" has thus been shown to be the binary mixture of the trans-trans-trans and the trans-cis-trans isomer.

105

ISOBACTERIOCHLORINOGEN / PORPHYRINOGEN - EQUILIBRIUM

SO' / 40 h =-95% ('H-NMR) 71,57, (crystallized)

R • CHjCHJ

No. 20

Figure 20: The mixture of the dipyrrolic dihydroderivatives of the t,t,t/ t,c,t-isobacteriochlorin pair (see above) is quantitatively converted into octaethylporphyrinogen by tautomerization with pyridine acetate. This observation illustrates clearly that in the absence of metal ions the tetrapyrrolic porphyrinogen structure is thermodynamically favored relative to isomers containing a partially corrinoid chromophore. Results on the chemistry of hexahydroporphinoids such as those described in Figures 16-20 can only be obtained when all experimental operations are carried out under exclusion of oxygen as strictly as possible (argon atmosphere in "drybox"). Experimentation in this field is particularly difficult and demanding; Christoph Angst, whose work this is, deserves to be specifically mentioned in this context.

106

C00H

COOH

SIROHYDROCHLORIN L.M. Siegel (1973) A.R. Battersby. VY Bykhovskii (1977) A.I.Scott

(1977/78)

ISOBACTERIOCHLORINS

No.21

FROM CORPHINS

Figures 21 and 22: The presence of isobacteriochlorins on the palette of ligand structures formed in our tautomerization experiments with octaethylporphinogen appears of special interest in the light of the structure and biological function of the newly dis18}

covered porphinoid natural product sirohydrochlorin

. At a

time, when the structure of this bacterial coenzyme ligand was not yet definite and its role in vitamin B

biosynthesis

107

not yet known. Professor Battersby proposed to settle its structure by synthesis using methods and intermediates available from the work on vitamin B ^

synthesis, and so we agreed

to attack this problem jointly. What was needed first was a rational de novo synthetic approach to the isobacteriochlorin ligand structure. Figure 23 shows a solution of this problem. (Besides Eisner's and Inhoffen's work on the reductive formation of isobacteriochlorin from porphyrins 16)17)^

oniy

the

pyrrolytic formation of a isobacteriochlorin complex from a 1 9 )

corresponding corphin complex

had been known at that time,

see Figure 22.)

DE NOVO S Y N T H E S I S OF

ISOBACTERIOCHIORINS

(«-TEMPLATE ,CH,

APPROACH)

a)

(CHj)JO* b) HjC=Cm

NalO, H' t • aQ*CHjOH H

. . / ~

C 0

'

C H

>

H'

carefully defined conditions afforded the methyl cyclopentene derivative which was oxidized with SeO~ in Ac„0 to afford the allylie acetate.

133

Peracid oxidation of the allylic acetate can be controlled to produce a single stereoisomer which vie suspect at this stage has the stereochemistryindicated. Nucleophilic opening with phenylselenol proceeded regioselectively to produce the corresponding selenide which suffered smooth elimination when treated with

to afford the enediol monoacetate. At the

time of this writing we anticipate the final conversion to the cyclopentenone oxide to proceed via the route indicated which was used so successfully in the C-ring series. Should this fail we would employ the alternative, chirally more dangerous, sequence developed for the A=B-rings.

To summarize our present position - we have new prepared the epaxyketones required for the A, B and C-rings and the D-ring is nearing completion and we are in the midst of producing these intermediates in quantity for the final assault. We have learned with experience that very little can be taken for granted in a project of this order of magnitude, and although vie feel we have done our "hanework" properly, there will undoubtedly be surprises and new challenges to be overcome. For example, at the tims of this writing we have started to examine the crucial Eschenmoser-Tanabe fragmentation of the C-ring epoxyketone. In spite of the fact that earlier model studies had been an unqualified success we have been unable

13i»

to effect this vital transformation in anything approaching a respectable yield. Our best yield to date has been 34% enploying the conditions indicated. This temporary frustration painfully illustrates the point that very little can be taken for granted and reminds us of the experimental nature of our science.

It remains for me to acknowledge with heartfelt thanks the splendid efforts of many skilled and dedicated coworkers. In addition to those names cited in the references below should be added Dr. John Chang, Dr. Harold Weller, and Dr. Rafael Shapiro, Dr. Steve Schow, and Dr. Marcus Schlageter. This work was supported financially by the National Science Foundation.

References 1. Stevens, R.V., Kaplan, M., "Studies on the Synthesis of Corrins and Related Ligands. A Simple Synthesis of y-Substituted Butyrolactams via the Conjugate Addition of Cyanide to aß-Unsaturated Ketones": Chem. Carm., 822-823 (1970). 2. Stevens, R.V., DuPree, L.E., Jr., Wentland, M.P., "Studies on the Synthesis of Corrins and Related Ligands. Employment of Isoxazoles as Intermediates in the Sythesis of Semicorrins": Chan. Cortm., 821822 (1970) . 3. Stevens, R.V., Christensen, C.G., Edmonson, W.L., Kaplan, M., Reid, E.B., Wentland, M.P., "Studies on the Synthesis of Corrins and Related

135

Ligands. I. General Approach and Model Studies,: J. Amar. Chan. Soc. 93, 6629-6635 (1971). 4. Stevens, R.V., DuPree, L.E., Jr., Edmonson, W.L., Magid, L.L., Wentland, M.P., "Studies on the Synthesis of Corrins and Related Ligands. II. The Hrploymsnt of Isoxazoles in the Synthesis of Semicorrins": J. Amer. Chem. Soc. 93, 6637 (1971). 5. Stevens, R.V., "Studies on the Synthesis of Corrins and Related Ligands. An Approach to the Total Synthesis of Vitamin B-12": Proc. 24th National Organic Syrrposium, 21-35 (1975). 6. Stevens, R.V., Christensen, C.G., Cory, R.M., Thorsett, E., "The Total Synthesis of Nickel(II)Octamethylcorphin": J. Amer. Chem. Soc. 97, 5940-5942 (1975) . 7. Stevens, R.V., Reid, E.B., "Studies on the Synthesis of Oorrins and Related Ligands. An Alternate Synthesis of "Semicorrin E": Tetrahedron Letters 48, 4193-4196 (1975). 8. Stevens, R.V., "Studies on the Synthesis of Corrins and Related Ligands": Tetrahedron 32, 1-14, (1976). 9. Stevens, R.V., Fitzpatrick, J.M., Germeraad, P.B., Harrison, B.L., LaPalme, R., "Studies on the Synthesis of Vitamin B-12. 1. Introduction and Model Studies": J. Amer. Chem. Soc. 98, 6313-6317 (1976). 10. Stevens, R.V., Cherpeck, R.E., Harrison, B.L., Lai, J., LaPalme, R., "Studies on the Synthesis of Vitamin B-12. 2. Synthesis of the "Southern Half": J. Amer. Chem. Soc. 98, 6317-6321 (1976). 11. Stevens, R.V., Gaeta, F.C.A., "Camphorae: Chiral Intermediates for the Total Synthesis of Steroids": J. Amer. Chem. Soc. 99^, 61056106 (1977).

NEW R E A C T I O N S OF

OF

THE

CHROMOPHORIC

VITAMIN B 1 2

SYSTEM

DERIVATIVES

H. H. Inhoffen Institut für O r g a n i s c h e C h e m i e Technische Universität Braunschweig;

D-3300

Braunschweig

Dicyano- 10-bromo-cobyrinicacid-heptamethylester m e t h y l a c e t a t e at - 7 8 ° C with /w 1 , 2 m o l ozone. ing up 46% of m o n o - s e c o - 5 ,

(2) is t r e a t e d in

After reductive work-

6-dioxo-dicyano-10-bromo-cobyrinicacid

- h e p t a m e t h y l e s t e r (3a) a r e i s o l a t e d .

A f t e r r e d u c t i o n of 3a with NaBH^ and following t r e a t m e n t with H^SO in CHgOH d i c y a n o - c o b y r i n i c a c i d - h e p t a m e t h y l e s t e r in 18% y i e l d .

© 1979 Walter de Gruyter & Co., Berlin • New York Vitamin BJ 2

(1) i s r e f o r m e d

13B

T r e a t m e n t of 3a with NaSH in p y r i d i n e / C H ^ O H and H^S y i e l d s a m i x t u r e of 13% d e s c o b a l t o - 1 0 - b r o m o - m o n o - s e c o - " c o b e s t e r " a s 46% of the 1 0 - H - a n a l o g u e

(4b).

4a

R = Br

(13%)

4b

R = H

(46%)

(4a) a s well

139 R e c o b a l t a t i o n of 4b with CoCl_ in CH_Cl_/CH_OH yields ( a f t e r c y a n i j \

—

2,

Z

Z

o

sation) 52% of 3b.

B r o m i n a t i o n with b r o m o - s u c c i n i m i d e f o r m s 3a,

which a f t e r r e c y c l i s a -

tion under known conditions f o r m s again 1 in 18% yield. All compounds a r e identified by a n a l y s e s , UV/ v i s - , I R - , 13 C-NMR, E I - and F D - M S , CD- and O R D - s p e c t r o s c o p y . I thank for c o l l a b o r a t i o n :

and

L . E r n s t , A. G o s s a u e r , K. - P . H e i s e ,

R . - P . Hinze, H. L a a s , H. M. S c h i e b e l , and H . - R .

1) throughout the reaction sequence

Schulten.

S T R U C T U R E A N D R E A C T I V I T Y O F THE S O - C A L L E D STABLE Y E L L O W

CORRINOIDS

B. GrC/ning and A . Gossauer Institut für O r g a n i s c h e Chemie der Technischen Universität Braunschweig, S c h l e i n i t z strasse, D - 3 3 0 0 Braunschweig, W . - G e r m a n y

Most reactions of cyanocobalamin (vitamin B ^ ) w h i c h proceed with alteration of the oxidation state of the complex-bounded cobalt ion are accompanied by the f o r mation of by-products w h i c h are called stable yellow corrinoids

. A m o n g the different

reactions w h i c h lead to the formation of stable yellow corrinoids, we have investigated first the formation of such pigments under the conditions of the Udenfriend reaction, bearing in mind that both vitamin B ^ coenzyme and ascorbic a c i d are natural c o m pounds w h i c h occur in the living c e l l . Two years a g o , the structure of the stable yellow corrinoid 2 w h i c h is formed by treatment of a solution of dicyanocobyrinic a c i d heptamethylester in aqueous methanol with ascorbic a c i d in the presence of o x y g e n was elucidated in our laboratories, and a reaction mechanism involving a 2 hydroxyl radical (or some equivalent I) was suggested reaction product revealed the presence of a hydroxy

. The X - r a y analysis of the group at C - 5 as well as of a

five-membered lactone ring between C - 6 and C - 7 .

CN

3

Present adress: Institut für Organische Chemie der Technischen Universität Berlin, Strasse des 17. J u n i 135, D - 1 0 0 0 Berlin 12, W . - G e r m a n y

© 1979 Walter de Gruyter & Co., Berlin • New York Vitamin B l 2

1 423-445 (1962). 5. Bonnett, R.: Chem. Rev. 63, 573-605 (1963). 6. Bernhauer, K., Müller, 0., Wagner, F.: Advanc. Enzymol. 26, 233-281 (1964). 7. Popova, Y., Ilieva, M.: Trav. Sei. Inst. Technol. Sup. Ind.Alim. Bulg. XIX, 246-247 (1972).

TEMPERATURE-JUMP KINETICS OF THE "BASE-ON"-"BASE-OFF" EQUILIBRIUM OF METHYLCOBALAMIN

K.L. Brown and A.W. Awtrey The University of Texas at Arlington Arlington, Texas

76019

USA

P.B. Chock and S.G. Rhee The National Heart, Lung, and Blood Institute Bethesda, Maryland

USA

Temperature-jump experiments have been performed on methylcobalamin in aqueous solution, ionic strength 1.0 M (KC1), at 5.0°, after a 5° C jump. At all pH's studied the kinetic transients showed two relaxations, which can be cleanly resolved from the heating rise-time (heating TJ 1.0 ys). The results (Table) show that the slower relaxation (t^"'') has a slight pH dependence with TJ ca. 50 ys at pH ca. 5.5-7.5, increasing to about 75 ys at pH 1.6 and below.

The faster relaxation

pendent over the range ca. 0.5-7.5 and had H

was apparently pH indeca. 4.4 ys.

Assuming that proton transfers to and from the benzimidazole moiety are diffusion controlled and hence much faster than either relaxation, the appearance of two relaxations means that at least three species are present in addition to the base-off benzimidazolium species of methylcobalamin.

It

can be shown [1] that a two-step reversible mechanism (eq. 1) will display two relaxations and that the reciprocal relaxation times are given by:

k2 t^1 V

1

K^ = k] + k2

(2)

= k 3 (l/(l + k g / k ^ ) + k 4

(3)

In the case of the methylcobalamin "base-on"-"base-off" equilibrium, two

© 1979 Walter de Gruyter & Co., Berlin • New York Vitamin B [2

zoo Table:

Reciprocal Relaxation Times for the Fast and Slow Relaxation of Methylcobalamin, 5.0° C, Ionic Strength 1.0 M

pH

T ^ 1 , s"1

0..46

5 1..49±0.,15xl0

0..97

1,.38±0. lOxlO

5

1..28

- •- -

- -- -

T 2 " \ S"1

- - -

N2a

3

9.,04±0.,54xl03

3

2

,92x10 9., 69±0.

3

3

-

9..40±1. OOxlO 3

2

8.,14±0.,38x10

3

4

4

4

1.,61

1..89±0.,10x10®

5..52

1..54±0.,06xl0

5

2

1..21+0.,07xl0

5.,95

1,.53±0.,15xl05

5

1.,38+0.,05xl04

3

6..47

1 .65±0.,06xl0

5

1

1.,42±0.,20xl0

4

3

7..42

5 1,.48±0.,24xl0

2

1..20±0..30x104

3

a

1

Number of traces analyzed

kinetically indistinguishable mechanisms of the type shown in eq. 1 can be written:

a limiting dissociative mechanism (D) in which an intermediate of

reduced coordination is formed, and a dissociative interchange mechanism (1^) in whijch an outer sphere complex is formed prior to ligand substitution [2],

For illustration, this analysis will assume a D mechanism (eq.

4) (analysis based on an CHo

CH 0

1 3

l-Co I 0H L

2 BZH +

mechanism would be strictly analogous). CH-j HÖH I3 k ™ " r Co off,

1 3

K„, Bz

rCo |

~±H+

2 Bz

I

' . HOH on

II

CH«

1 3

Bz k„

Co rCo

r

Bz

kBz off

III

K. L

K C-W

fez

^

IV

The microscopic equilibrium constants are then given by: K l = [IV]/[III] =

(5)

K c . w = [iii]/[n] = k ™ x 2

H

K B z = [H][H+]/[I]

(6) (7)

Two macroscopic equilibrium constants may also be defined (eqs. 8 and 9). K g = ([II] + [III] + [IV])[H + ]/[I]

(8)

K f = [IV]/([II] + [III])

(9)

By making the appropriate substitutions from eqs. 5-7 one obtains: K

a =

+

K

c-w

+

K

C-W K L) K BZ

201

K

K

f =

LKCV(KC-W+

We have tentatively assigned the faster relaxation of methylcobalamin to the water on-off equilibrium (K c -1 T

=

1

in eq. 4) and hence, from eqs. 1-4: kHOH

.HOH

off

,

.

on

To substantiate this assignment, we performed kinetic measurements on the temperature dependent spectral changes of methyl- and ethyl(aquo)cobaloxime which are thought to be due to perturbation of the water on-off equilibrium [3].

A single relaxation was observed (0^-5° jump, pH 6.48) with Ti 14.7 ys

for methyl- and 11.4 ps for ethyl(aquo)cobaloxime in good agreement with Ti for the faster methylcobalamin relaxation.

This assignment is also in

agreement with the lack of pH dependence for the faster relaxation.

Tem-

perature-jump experiments with methylcobinamide are in progress to substantiate this assignment and discriminate between the D and

mechanisms.

If this assignment is correct, the slower relaxation of methylcobalamin must be due to benzimidazole coordination and V

1

= ( K Bz/(£H + ] + K

B Z

))(

W

(K

c

_

w

+

l)) k ;; + k £ f

(13)

where Kg z /([H + ] + Kg z ) accounts for the inability of protonated benzimidazole to contribute to the rate of coordination. Consequently when T

and when [H

[H+]»Kgz

-1 _ i,Bz K off U 2(acid)

(14)

]«KR

Bz 1 From the data we calculate as the average of at pH 1.6 and below o 1 (9.07±0.70xl0 s average of 12 traces). From this value, eq. 15 and the average of

-1

/ 4 -1 at pH 5.5 and above (1.30±0.11x10 s average of 13

traces) we calculate kj^

K

c.w/^

K

c-w

+

1

)

to

be

3.9xl0 3 s" 1 .

This allows

calculation of the macroscopic formation constant, K^, from eqs. 5, 9 and 11.

The value obtained for K^ is 0.43 which leads to the startling con-

clusion that the "base-on" species of methylcobalamin (i.e. methylcobalamin above pH 5.0) is only about 30% coordinated to benzimidazole, the remainder

202

being d i s t r i b u t e d between species I I and I I I We can now make certain assumptions on K

•

(eq. 4). I f the intermediate of r e -

duced coordination (species I I I , eq. 4) i s formed only as a t r a n s i e n t intermediate and does not contribute s i g n i f i c a n t l y to the equilibrium d i s t r i b u t i o n of species above pH 5 ( i . e . K C _ W

+

K p ì

fa ~ co

£ m .e o

, 1 • ' a ***

«G •C ^ „ 7 „ t-J M J W

« o

° o i

« <

3

3

?

H:UC

M " ' \ - C O

Ratio 126

Scheme 7. Enzymic conversion of doubly-labelled trimethylisobacteriochlorin into cobyrinic acid.

2

H

234 stage

during the sequence o f

acid

(31).

This

finding

C-20;

raises

interesting

l o s s might occur

the nature reported

of

reactions

which produce

questions

loss

a s a C 2 ~ u n i t o r a s two C ^ - u n i t s

the extruded fragment i s

(ref.32)

about the

cobyrinic

t o be

lost

as

of

and

being studied £c-20

is

formaldehyde^ .

The p o i n t h a s now b e e n r e a c h e d w h e r e t h e

following

biosynthetic

sequence has been e s t a b l i s h e d

f o r t h e pathway t o v i t a m i n

B^

(1):-

( 4 ) —>• p o r p h o b i l i n o g e n

—

S-Aminolaevulinic

uro'gen-III into

dihydrosirohydrochlorin

Further methylation (16)

yields

(15^)

vitamin

B^

be b u i l t

be given

into

acid the

a t t h e end o f

" I n w h a t o r d e r a r e r i n g s A, B ,

to the early focussing

steps

This in the

attention

uro'gen-III

on t h e

is

best

foregoing

reaction

conversion

o f PBG

question

vitamin back

sequence (¿)

final

this

answered by l o o k i n g

shown i n C a m b r i d g e o v e r t h e

t h a t when t h e two e n z y m e s d e a m i n a s e together to build uro'gen-III r i n g - A and C - 2 0 , derived

ring-B

from i n t a c t

ring-D undergoes

rearranging

carbon

(8)

and

into

and C - 5 ,

last

and c o s y n t h e t a s e

intramolecular

in t h i s

(19),

formed by h e a d - t o - t a i l

units;

the

are

(.8)

pro-

rearrangement,

with

(33).

p r o c e s s was shown t o b e t h e

unrearranged f o u r PBG

amino g r o u p o f

m i g h t b e d e r i v e d f r o m some o t h e r Double

the

A key

assembly of the

group X c o u l d be t h e o r i g i n a l

f r o m a g r o u p on t h e e n z y m e .

(S) ,

t h e PBG u n i t

forming C-15

intermediate

(15)

work

ring-C with C-10 of

PBG u n i t s w h i l s t

atom f i n a l l y

ten years

f r o m f o u r PBG u n i t s

bilane

unit or i t

C and D o f

(2)->

(8).

has been

perhaps

(18) — » c o b y r i n i c

T h i s knowledge w i l l

assembled"?

viding

(14) .

B u t f i r s t we c a n c o n s i d e r o n e v e r y i n t r i g i n g

which i s :

all

(5)

converted

dihydrotrimethylisobacteriochlorin

t h e pathway which w i l l

lecture. b12

(¿)•

is

sirohydrochlorin

trimethylisobacteriochlorin

map o f

It

acid

(J5) w h i c h w i t h S - a d e n o s y l m e t h i o n i n e

a PBG

nucleophile, 13 C-labelling

235

C0 2 H.

Enzyme Deaminase cosynthetase

(19) Bilane

Scheme

8.

A = CH 2 C0 2 H

P = CH 2 CH 2 C0 2 H

experiments based on synthetic samples of the bilane ( — , X = + NH^) , as illustrated in Scheme 8, proved that deaminase-cosynthetase convert the unrearranged bilane system (19^) into uro'gen-III

(.8) .

The carbon atoms labelled at 9

with

13

C

were shown to become intramolecularly bonded and, in a separate experiment, those labelled at A together intramolecularly

(34,35).

sise that the rings of the bilane uro'gen-III

were also proved to join It is important to empha-

(19^) register with those of

(J3) as drawn in Scheme 8; that is, ring-A of the

bilane becomes ring-A of uro'gen-III and similarly for the other rings. It is remarkable that when deaminase alone (no cosynthetase) acts on PBG (_5) , the unrearranged uro'gen-I

(20) is formed;

importantly, this is not converted into uro'gen-III

(8)

by

cosynthetase nor by deaminase-cosynthetase acting together (2, 15).

In view of this, it was fascinating to find (36) that

when the synthetic bilane (19^, X = NH-,) was treated with

236

Figure 6. Enzymic formation of uro'gen macrocycl^ from the unrearranged bilane (19, X = NH^) with deaminase alone and with deaminase-cosynthetase together deaminase alone, there was a clear lag in the formation of uro'gen-I (20), yet there was no lag in the formation of uro'gen-III (ji) when the bilane with deaminase-cosynthetase

X = NH^) was treated

(Figure 6).

This lag was even

more pronounced when consumption of PBG (5^) by deaminase alone relative to production of uro'gen-I (20) was determined (Figure 7); again, there was no lag when deaminase and cosynthetase were used together.

The lag for deaminase alone

indicated that an intermediate was being released into the medium. The structure of the substance being produced by deaminase was established by (a) showing that it ring-closed chemically to 99% pure uro'gen-I (20); (b) generation of the substance from 11-

C PBG (as 5^) and stopping the enzymic reaction by adjust13 ment to pH >12 when ca. 80% of the original C-PBG had been 13 1 consumed; C-n.m.r. of the reaction mixture, with H-noise decoupling, gave Figure 8, whereas with off-resonance decoupling, all four signals appeared as triplets (so all are 13 from Ci^d). The integral for signal P: signal Q was 1:3

237

5

10

15

Time (mins) Figure 7. Enzymic formation of hydroxymethylbilane (19, X = OH) followed by ring-closure: A, without acided enzyme; B, with additional deaminase; C, with added deaminase-cosynthetase.

and the assignments under Figure 8 are based unambiguously on synthetic bilanes and hydroxymethylpyrroles.

The conclusion

was thus clear (36); the substance released into the medium as deaminase acts on PBG is the hydroxymethylbilaneI (19^ X = OH).

ishortly before our proof of this structure, Prof. A.I. Scott sent us preprints (37) reporting that deaminase acts on PBG to produce a cyclic substance, pre-urogen, of structure (2_1) . We feel that the evidence does' not support the existence of pre-urogen (2_1) but it strongly indicates that tITe Texas group is handling what is proved above to be the hydroxymethylbilane (19, X = OH).

P

A

'A

p

(11)

238

Q

Deaminase acting on

Figure 8. 1H-Noise decoupled 13C-n.m.r. spectrum of product from action of deaminase on ll_13c PBG. Signal P HOCH2 pyrrole, 65 7.21; Q, bilane CH2 (pyrrole)2, 624.46; R, H 2 NCH 2 of residual PBG, 638. 35; S, uro1 gen CH 2 (pyrrole)2, 623.97. All 6 referred to Me 3 SiCD2CD 2 C0 2 Na. This finding then allowed experiments leading to a fuller understanding of the enzymes involved in these early stages of vitamin B ^ biosynthesis (36). Thus, a further sample of hydroxymethylbilane (1£, X = OH), generated as above, was allowed from point A, Figure 7, to ring-close chemically to uro'gen-I (20) and a second equivalent portion was treated at point B, Figure 7, with additional deaminase; the two rates of formation of uro'gen-I (20) were essentially the same. So deaminase is not an enzyme for ring-closure; its function is to assemble from four PBG units the unrearranged linear tetrapyrrole system which appears in the medium as hydroxymethylbilane (19^, X = OH) . In striking contrast, when deaminase-cosynthetase was added to a third portion of hydroxymethylbilane at point C, Figure 7, almost instantaneous ring-closure occurred with rearrangement, to form

239

uro'gen-III (8).

Current work with pure cosynthetase will

complete the picture. One final point needs to be added concerning the formation of the unrearranged bilane system.

Not only does deaminase con-

vert PBG (5) into the hydroxymethylbilane

(19_, X = OH) but it

also forms this same hydroxymethyl system when it acts on + synthetic aminomethylbilane ( 1 9 X = NH^) , (38). Thus, the latter enters the normal biosynthetic pathway to uro'gen-III (8) at an advanced stage by enzymic replacement of the amino group, a possibility which had been emphasised earlier (2,15, 35,36) The foundation has now been laid to allow an answer to the question of order of assembly of the four PBG units of uro' gen-III (8) and so of vitamin B.^ (1).

The first lead came

from enzymic experiments with a set of synthetic isomers of the bilane (¿9, X = NH^), in which the order of the A and P groups was varied (39). These studies suggested that there are four binding sites on deaminase which recognise the four pyrrolic rings. These can be illustrated as empty sites A, B, C and D on the left of Figure 9. A considerable quantity of deaminase-cosynthetase was isolated to allow a deficiency 12

of normal

C-PBG (5) (2 equivalent) to be added (39).

After a short time at 4°C, the loading of the site onto which

15

Then chase

A

through with

Figure 9

'3

P

2hO

the first PBG unit binds should be highest, whilst the corresponding loadings for the other sites should decrease such that the last site to be filled carries least, as indicated on the right of Figure 9.

The loadings referred to cover

all pyrrolic material on that site whether it be monopyrrole or a part of a di-, tri, or tetra-pyrrole species. The partially loaded enzyme was then treated with an excess of 90 atom %

PBG (as 5_) to chase the bound pyrrolic

species through to form uro'gen-III (.8), see Scheme 9.

This

was dehydrogenated to uroporphyrin-IIl, and after chemical decarboxylation and esterification, the resultant coproporphyrin-III ester (22) was studied by

H-n.m.r. using Euifod)^

shift reagent.

C o p r o p o r p h y r i n - III (!)

t e t r a m e t h y l ester

Scheme

(|2)

9

The spectrum (Figure 10) was a joy to see since it showed that the

12

C-content

is greatest at C-20 and that the

12

C-levels

at the other bridges fall in the order C-5 > C-10 > C-15. Thus the first PBG unit to bind becomes ring-A (with C-20), the second forms ring-B (with C-5), the third ring-C (and C-10) and the fourth ring-D (and C-15). This clear result (38) comes 12 13 from using C-"labelling" against a background of C-material and some thought will bring out the advantages of this approach to this problem.

With

the

we c a n

intriging

summarise

which has been derived with

question

in

first

ment p r o c e s s methionine

is

sirohydrochlorin^ methylation The

then

subsequent

can

(1£) t * produces

conversion

C-methyl

in

the

(J3)

of

the

trimethyl (16^)

into

leading (14).

analogues cobyrinic

the

medium

presence

sirohydrochlorin the

lecture (5),

starting

produce

t o which

groups

answered,

this

assembled to

b y an i n t r a m o l e c u l a r

uro'gen-III

donates

are

last, In

in

F o u r PBG u n i t s

appear

X = OH).

ring-closed

t o produce

(SAM)

a s s e m b l y now

out.

(£),

ring-D

(lj)) , which (19^

of

carry

ALA m o l e c u l e s

rearranged bilane this

order

to

and a d d i n g

hydroxymethylbilane thetase,

of

10 t h e work o u t l i n e d

so e n j o y a b l e

from e i g h t

ring-A

Scheme

of

un-

as cosyn-

rearrangeS-adenosylto

dihydro-

Further (18) .

(16) acid

(2)

H-5 H-15

14

H-10

12

H-20

10

1. Figure 10. H-Nmr s i g n a l s f r o m t h e b r i d g e s (positions 5, 10, 15, 20) o f c o p r o p o r p h y r i n - I I I t e t r a m e t h y l e s t e r (22) i n p r e s e n c e o f E u ( f o d ) . , .

t.

For information about the stage of monomethylation, see 2 5 a n d t h e c o n t r i b u t i o n b y G. M t l l l e r i n t h i s v o l u m e .

ref.

242

P

Scheme 10. Map showing e x p l o r e d o f t h e pathway t o v i t a m i n B , 0 .

A

region

21+3 occurs with loss of C-20 and of the C-20 methyl group and finally cobyrinic acid B12

is elaborated to produce vitamin

(1).

Much remains to be discovered but if one stands back to look at the fascinating pathway to vitamin B ^ summarised above with all its twists and turns, it might reasonably be felt that the work has reached at least the end of the beginning. My feeling is that we are now starting out on the beginning of the end.

Acknowledgements Progress in this field at Cambridge has depended on the contributions of a succession of outstanding young colleagues. All their names are given in the references to the literature and the most recent work covered more fully in this lecture has been carried out by C.J.R. Fookes, N.G. Lewis, G.W.J. Matcham, M.J. Meegan, R. Neier, K.E. Gustafson-Potter, M. Thompson and W.-D. Woggon.

I am deeply indebted to them all.

I also wish to record my warmest appreciation to my senior colleague Dr. Edward McDonald who has been a tower of strength throughout.

Finally the financial support of the Nuffield

Foundation, S.R.C. and Roche Products Ltd is gratefully acknowledged.

References 1. 2.

Bernhauer, K., Wagner, F., Michna, H., Rapp, P., Vogelmann, H.: Hoppe Seyler's Z. Physiol. Chem. 349, 129 7 (1968). Reviewed by Battersby, A.R., McDonald, E.: Porphyrins and Metalloporphyrins, 2nd ed., p. 61. Ed. Smith, K.M. Elsevier, Amsterdam, 1975.

2bU 3.

Brown, C . E . , Shemin, D . , K a t z , J . J . : J. B i o l . Chem. 248, 8015 (1973) and r e f e r e n c e s t h e r e i n .

4.

S c o t t , A . I . , Townsend, C . A . , Okada, K . , K a j i w a r a , M., Whitman, P . J . , Cushley, R . J . : J. Amer. Chem. Soc. 94, 8267 (1972); and 96, 8069 (1974); S c o t t , A . I . , Georgopapadakou, N . , Ho, K . S . , K l i o z e , S . , L e e , E . , Temme I I I , G.H., Townsend, C . A . , A r m i t a g e , I . A . : J. Amer. Chem. Soc. 9J_, 2548 (1975) and r e f e r e n c e s t h e r e i n ; Reviewed by S c o t t , A . I . : Accounts Chem. Research 1]., 29, (1978).

5.

I n f i e l d , M., Townsend, C . A . , A r i g o n i , D . : J . Chem. Soc. Chem. Comm. 541, (1976).

6.

B a t t e r s b y , A . R . , I h a r a , M., McDonald, E . , Stephenson, J . R . : J. Chem. Soc. Chem. Comm. 404, (1973); B a t t e r s b y , A . R . I h a r a , M., McDonald, E . , Stephenson, J . R . : J. Chem. Soc. Chem. Comm. 458, (1974); B a t t e r s b y , A . R . , I h a r a , M., McDonald, E . , Satoh, F . , W i l l i a m s , D . C . : J. Chem. Soc. Chem. Comm. 4 36, (19 7 5 ) .

7.

a) B a t t e r s b y , A . R . , I h a r a , M., McDonald, E . , R e d f e r n Stephenson, J . R . , G o l d i n g , B . T . : J. Chem. Soc. Perkin I , 158, (1977). b) B a t t e r s b y , A . R . , McDonald, E . , H o l l e n s t e i n , R . , I h a r a , M., Satoh, F . , W i l l i a m s , D . C . : J. Chem. Soc. Perkin I , 166, (1977) and r e f e r e n c e s t h e r e i n .

8.

Keese, R . , Werthemann, L. , Eschenmoser, A . : unpublished work: c f . Werthemann, L . , D i s s . No. 4097, E.T.H. Z u r i c h , 1968.

9.

B a t t e r s b y , A . R . , I h a r a , M., McDonald, E . , Stephenson, J . R . , G o l d i n g , B . T . : J. Chem. Soc. Chem. Comm. 458, (1974).

10.

O z o n o l y s i s and i s o l a t i o n o f c r y s t a l l i n e r i n g C i m i d e , Bogard, T . L . , Eschenmoser, A . : unpublished work.

11.

Dubs, P . , Eschenmoser, A . : unpublished r e s u l t s : Dubs, P . : D i s s . No. 4297, E.T.H. Z u r i c h , 1969.

12.

S c o t t , A . I . , Townsend, C . A . , Cushley, R . J . : J. Amer. Chem. Soc. 95, 5759, (1973).

13.

B a t t e r s b y , A . R . , H o l l e n s t e i n , R . , McDonald, E . , D . C . : J. Chem. Soc. Chem. Comm. 543, (1976).

14.

S c o t t , A . I . , K a j i w a r a , M., Takahashi, T . , A r m i t a g e , I . M . , Demon, P . , P e t r o c i n e , D.: J. Chem. Soc. Chem. Comm. 544, (1976).

15.

Reviewed by B a t t e r s b y , A . R . , McDonald, E . : Accounts Chem. Research. 12, 14, (1979).

16.

Dauner, H . - O . , M i l l i e r , G. , Hoppe S e y l e r ' s Z. Chem. 356, 1353 (1975).

17.

Reviewed by B a t t e r s b y , A . R . , McDonald, E . : B i o o r g . Chem. 7, 161, (1978).

cf.

Williams,

Physiol.

21*5 18.

Bykhovsky, V. Ya., Zaitseva, N.I., Bukin, N.V., Doklady Acad. Sei. USSR. 224, 1431 (1975): Bykhovsky, V. Ya., Zaitseva, N.I., Umrikhina, A.V., Yavorskaya, A.N., Priklad. Biokhim. Mikrobiol. 12. 825 (1976); Bykhovsky, V. Ya., Zaitseva, N.I.: Priklad Biokhim. Microbiol. 12, 365 (1976).

19.

Eisner, U., J. Chem. Soc. 3461 (1957). Bonnett, R., Gale, A.D., Stephenson, G.F.: J. Chem. Soc. C, 1168, (1967). Whitlock, H., Hanauer, R., Oester, M.Y., Bower, B.K.: J. Amer. Chem. Soc. 91, 7485, (1969); Inhoffen, H.-H., Buchler, J.W., Thomas R.: Tetrahedron Lett. 1141, (1969). Battersby, A.R., McDonald, E., Morris, H., Thompson, M., Williams, D.C., Bykhovsky, V. , Zaitseva, N., Bukin, V. : Tetrahedron Lett. 2217, (1977).

20. 21.

22. 23. 24.

Siegel, L.M., Murphy, M.J., Kamin, H.: J. Biol. Chem. 248, 251, (19 73); Murphy, M.J., Siegel, L.M.: J. Biol. Chem. 248, 6911 (1973); Murphy, M.J., Siegel, L.M., Kamin, H., Rosenthal, D.: J. Biol. Chem. 248, 2801 (1973). Battersby, A.R., Jones, K., McDonald, E., Robinson, J.A., Morris, H.R.: Tetrahedron Lett. 2213, (1977). Battersby, A.R., McDonald, E., Thompson, M., Bykhovsky, V. Ya.: J. Chem. Soc. Chem. Comm. 150, (1978). Battersby, A.R., Haslinger, E., McDonald, E., Robinson, J.A., Thompson, M.: unpublished work.

25.

a) Deeg, R., Kriemler, H.-P., Bergmann, K.-H., Müller, G.: Z. Physiol. Chem. 35 8, 339, (1977); b) Bergmann, K.-H., Deeg, R., Gneuss, K.D., Kriemler, H.-P., Muller, G.: Z. Physiol. Chem. 358, 1315, (1977).

26.

Arigoni, D., Imfeld, M.: unpublished work.

27.

Battersby, A.R.: Experientia, 34' 1/ (1978).

28.

Scott, A.I., Irwin, A.J., Siegel, L.M., Shoolery, J.M.: J. Amer. Chem. Soc. 100, 316 and 7987, (1978).

29.

Professor A. Eschenmoser's group (E.T.H., Zürich) is studying the chemistry of simpler dihydroisobacteriochlorins (see his contribution to this volume) and we are grateful for valuable discussions with Professor Eschenmoser. Battersby, A.R., Matcham, G.W.J., McDonald, E., Neier, R., Thompson, M., Woggon, W.-D., Bykhovsky, V. Ya., Morris, H.R.: J. Chem. Soc. Chem. Comm. 185, (1979).

30. 31.

Lewis, N.G., Neier, R., McDonald, E., Battersby, A.R.: in press.

32.

Kajiwara, M., Ho, K.S., Klein, H., Scott, A.I., Gossauer, A., Engel, J., Neumann, E., Zilch, H.: Bioorganic Chemistry 6, 397, (1977).

2k&

33.

Battersby, A.R., Hunt, E., McDonald, E. : J. Chem. Soc. Chem. Comm. 442, (1973); Battersby, A.R., Hodgson, G.L., Hunt, E., McDonald, E., Saunders, J.: J. Chem. Soc. Perkin I, 273, (1976).

34.

Battersby, A.R., McDonald, E., Williams, D.C., Würziger, H.K.W.: J. Chem. Soc. Chem. Comm. 113, (1977); cf. Dauner, H.O., Gunzer, G., Heger, I., Müller, G.: Z. Physiol. Chem. 35 7, 147 (1976) for work in the unlabelled series.

35.

Battersby, A.R., Fookes, C.J.R., McDonald, E., Meegan, M.J.: J. Chem. Soc. Chem. Comm. 185, (1978).

36.

Battersby, A.R., Fookes, C.J.R., Matcham, G.W.J., McDonald, E., and (in part) Gustafson-Potter, K.E.: J. Chem. Soc. Chem. Comm. (1979) in press.

37.

Burton, G., Fagerness, P.E., Hosozawa, S., Jordan, P.M., Scott, A.I.: J. Chem. Soc. Chem. Comm. (1979) in press.

38.

Battersby, A.R., McDonald, E.: J. Battersby, A.R., McDonald, E.: J.

39.

Fookes, C.J.R., Matcham, G.W.J., Chem. Soc. Chem. Comm. (1979) in press. Fookes, C.J.R., Matcham, G.W.J., Chem. Soc. Chem. Comm. 1064, (1978).

INTERMEDIARY METABOLISM OF COBYRINIC ACID BIOSYNTHESIS

A. I . Scott Department of Chemistry, Texas A&M University, College Station, Texas 77843, U.S.A.

Introduction The discovery (1) that corrins, like heme and the chlorophylls, are formed in Nature from 6-aminolevulinic acid (ALA, 1_) and, most probably, porphobilinogen (PBG, 2) was made over twenty years ago and finds mention in the Second Symposium on Vitamin B ^ (2).

The important observations of

Bernhauer (3) which link cobyrinic acid (3aJ to the vitamin

(as cyano-

cobalamin 3cJ and coenzyme f i l l e d a considerable void in the metabolic sequence but s t i l l l e f t uncharted the territory between PBG and cobyrinic acid. by

In contrast to the varied and i n d i v i d u a l i s t i c strategies employed

our colleagues intent on the chemical synthesis of corrins

Chapter

1

(see

), which can be compared with the ascent of a mountain by

alternative routes, the biosynthesis of vitamin B ^ has already evolved as a unique, dynamic function of the c e l l , whose intricate details await elucidation.

Thus, the biosynthetic challenge finds analogy in the d i s -

covery of an ancient architectural masterpiece built from familiar materials but by unknown techniques of construction.

The problem i s one

of careful reconstruction of the design and execution of each stage, including periods where parts of the original structure have been removed and later restored in a different style.

Many intricate facets remain to

be established before the complete plan of the master builder emerges, but the principal features have recently become d i s t i n c t in a most s a t i s f y i n g and quite surprising manner. Armed with the knowledge that the corrin nucleus i s constructed from the building blocks of ALA, methionine and cobalt ion, we began, in 1970, the microbiological, analytical and synthetic experiments necessary for the

© 1979 Walter de Gruyter & Co., Berlin • New York Vitamin B 12

2kB

observation of regiospecifically enriched carbons in the CMR spectra of corrins biosynthetically derived from Propionibacterium shermanii.

As the

account unfolds, we shall have occasion to allude to the timely contributions made by other teams of explorers in search of intermediates and their biochemical connections.

We f i r s t review experiments designed to establish

both the number and the mode of insertion of the methionine-derived methyl groups and of the ALA molecule into the corrin nucleus as a background for the development of the enzymology of cobyrinic acid synthesis, including a brief glimpse of some novel probes for the enzymes at work on corrin construction.

F i n a l l y , we discuss the isolation and structures of new inter-

mediates and their implication for the mechanism of B ^ formation.

A.

The Carbon Balance and the Origin of the Methyl Groups in Vitamin B ^

Of the eight methyl groups attached to the periphery of (3c) i t was suggested (4) that those at C-l and C-12 stem from C-5 and C-2 of ALA, respectively, the latter by a well documented decarboxylation of acetate attached to the uro'gen system, while the derivation of the former (C-l) methyl group could be envisioned either as a reduction of a CH2 bridge of uro'gen I I I (4), or as a result of direct cyclization of a linear tetrapyrrole (5), the s i x remaining methyl groups a r i s i n g from methionine. Support for these ideas came from Kuhn-Roth oxidation of corrinoids labeled with [ 5 - 1 4 C ] - and [2,3- 1 4 C]-ALA and [ 14 CH 3 ]-methionine (4). When the problem was reexamined using

13 C Fourier transform NMR, adminis-

tration of [2- 1 3 C]-ALA to P. shermanii (ATCC 9614) afforded a sample of cyanocobalamin (3c) in which eight high-field signals in the 13 CH~ and CH, region were enriched (6a-c) in the proton noise-decoupled C FT-NMR 13 spectrum. Assignments of the eight C resonances were made to the seven CHpCONH, methylenes and one of the gem-dimethyl groups of ring C in full 14 accord with earlier C studies (see Figure 1 and Table 1). A sample of 13 B-|2 enriched by feeding [5- C]-ALA provided the surprising result that, of the eight anticipated enriched carbons, only seven signals appeared in 2 the low f i e l d region associated with sp (C = C and C = N) functions. The s p l i t t i n g pattern predicted for the distribution of label i s i l l u s t r a t e d

ZkS

^c-s-ch2ch^chcooh

COOH

< • '

*

>cOOR

(3a)

R=R'=H

(3b)

0R=NH 2 ; 0R'=NHCH 2 CH(CH 3 )0H

(3c)

0R=NH2; OR'=aminoisopropanolphosphate-ribose5,6-dimethylbenzimidazol e

(3d)

as in 3b except ring C: . JS

(3e)

R=R'=CH 3

Co -CN

OOR

COOH COOH HOOC,

HOOC, COOH

COOH HOOC

HOOG

FIGURE 1

25G

TABLE 1.

C Chemical shifts 3 for cyanocobalamin and dicyanocobalamin with proton decoupling (6 from HMDS) based on enrichment data for 26 carbon atoms.

Enriched Position^

Cyanocobalamin

Dicyanocobalamin

C Methyl at C 1 2 (pro-S)

•

32.4

31.6

C Methyl at C ] 2 (pro-R)

*

20.1

19.6

C Methyl at C 5 and C 1 5

*

16.2, 16.5

15.8, 16.3

C Methyl at C-|, C 2 , C 7 , C-,7 *

16.9, 17.9 20.5, 20.4

17.5, 18.4 19.7, 22.7

Propionamide a-methyl enes (Oi 2 CONH 2 ) at C 3 , C 8 , C 1 3 , C ] 7

•

32.4, 32.8, 33.6 35.7 or 36.0

32.9, 33.0 33.2, 33.5

Acetamide a-methylenes (CH 2 C0NH 2 ) at C 1 8 , C 2 , C y

•

35.7 or 36.0 43.9, 44.1

36.1 43.3, 44.9

Propionamide 3-methylenes (•i 2 CH 2 C0NH 2 ) at C 3 , Cg, C 1 3

•

27.0(2C), 29.1

25.9, 26.8, 27.8

Propionamide ß-methylene at C 1 7 • C

5

•

C

10

•

C

15

C

" C

4' 16 C9

c14 a

B

33.3

34.1

108.6

106.3

96.0

92.2

105.2

104.2

180.1 , 181.1

179.6, 179.7

•

174.6

173.1

•

166.4

164.1

In ppm (± 0.2 ppm). These values correct earlier data (6) which contain a computer-derived error. The assignments are unchanged.

^See Figure 1 for a sutimary of the enrichment scheme and the numbering system.

in Figure 1.

Such an array is in harmony with current ideas on the mecha-

nism of type III uro'gen formation (vide infra) and this result (6) was simultaneously and independently confirmed in Shemin's laboratory (7) in 13 1972. However, there was no C-enhanced signal above 95 ppm downfield from HMDS showing that no enrichment of the C-l methyl occurred. This 13 indicates that one of the CH 2 NH 2 termini of ALA (and hence of PBG or uro'gen III) has been extruded in the formation of the vitamin.

The origin

of the "missing" C-l methyl group turned out to be methionine (6a). The 13 13 C FT spectrum of dicyanocobalamin obtained by feeding [ CHo]-methionine

251

revealed seven signals highly enriched above natural abundance (C* in Figure 1) between 15 and 23 ppm (Table 1), a result which was to receive confirmation from the work of the Cambridge group (8) in which the use of cobester (3e) simplified the CMR analysis.

B.

Stereochemistry of Methyl Group Insertion in Corrinoid Biosynthesis

Before developing further mechanistic proposals for corrin biosynthesis, which appears to be controlled by both steric and electronic consequences of methyl group insertion via S-adenosylmethionine (SAM), resolution of the problem of the stereochemistry of methylation at C-12 in ring C became necessary.

Although i t had been rigorously demonstrated that one of the

methyl groups at C-12 is derived from methionine and the other from C-2 of ALA (1_), the stereospecificity of this process had not been established. The following experiments provided a solution to this problem and demon13 strate the advantage of C-enriched shifts for the determination of carbon isotope c h i r a l i t y . Labeled specimens of dicyanocobinamide (3b) and neocobinamide (3dJ were obtained, where one of the C-12 methyl groups was s p e c i f i c a l l y enriched, 13 by feeding [ CH3]-methionine to P. shermanii c e l l s followed by hydrolysis (CFgC02H) and separation (9,10).

If the methionine-derived methyl at C-12

is a-oriented in the neo series, i t w i l l bear an anti-periplanar relationship to the propionamide side chain and the concomitant removal of the gamma effect should result in a downfield s h i f t of the enriched methyl resonance signal. This was clearly shown by the downfield s h i f t of ca^. 13 12 ppm in the

C FT-NMR spectrum for one of the methyl resonance lines

in going from cobinamide to neocobinamide (11). 1o These results established that the [ CH3]-methionine methyl (*) is inserted into the corrin nucleus at C-12 from the a-face 1 3 and that the absolute configuration at C-12 is (R). Furthermore, the C results rationalize the apparent anomaly (observed previously) that the g-methyl group (6) of the gem-dimethyl grouping at C-12, derived from C-2 of ALA (1_), resonates at substantially lower f i e l d (31.6 ppm) than the methyl region tentatively

252 assigned by Doddrell and Allerhand (12). 3 the remaining methyl groups at sp

It should be noted that all of

carbons appear at higher field (15-23

ppm), i.e. within that region proposed by Doddrell and Allerhand, because of gamma interactions.

An independent, different proof of the absolute

stereochemistry of the methylation at C-12 was obtained at Cambridge (13). At this stage of the investigation (1972-1973) the technique of cell-free corrin biosynthesis with P_. shermanii was developed at Yale (14) and at Cambridge (15).

The supernatant fraction (100,000 g) with appropriate

additives (14,15) is capable of transforming ALA, methionine, PBG and, as described below, uro'gen III to cobyrinic acid (3aJ.

A similar system

from Clostridium tetanomorphum which makes cobyrinic acid, but not heme, was developed simultaneously by Miiller (16).

C.

Concerning the Fate of the Methyl Group Protons

Early work at Yale (17) and at Cambridge (13) indicated that virtually intact methyl transfer from S-adenosylmethionine was taking place. transpires that in developing a theory for the mechanism of A

It

D ring

fusion the fate of the original protons of methionine assumes an important role with respect to the C-l methyl group. 13

2

C H 3 -methionine

(90% in

13

Therefore, a sample of

2

C ; 98% in H ) was administered to resting

whole cells of P. shermanii and the resultant purified cyanocobalamin (3c) examined by a technique developed in our laboratory to provide maximum 2 1 sensitivity for studying H- H exchange phenomena. As shown in Figure 2a the undecoupled ^H FT spectrum of the enriched sample 1 13 does not reveal any unusual H- C coupling of the methyl signal centered at 0.47 ppm, which has been unambiguously assigned to C-l (18,19). That 1 13 H- C coupling was present and not due to any exchange of the enriched 13 C-l methyl carbon was revealed by simultaneous subtraction of the Cdecoupled spectrum to give the difference spectrum shown in Figure 2b. The latter technique reveals that (a) all of the observable satellite peak intensity in Figure 2a is due to spinning side bands, (b) the ratio of 13 1 C-satellite to

H-resonance intensity at the C-l methyl (1/200 for each

satellite) (Figure 2b) is within experimental error (10%) identical with

253 natural abundance (1.1%) ( J 1 H - 1 3 C = 125 Hz), (c) the analysis of the difference spectrum corresponding to the chemical shifts for the C-2a, 13 C-12a and C-173 methyls (19) shows no exchange of these CDg groups (natural abundance satellites only) thus confirming an earlier conclusion (13), (d) a strong 3-bond coupling (J = 8.5 Hz) centered at 1.26 ppm due 13 1 to C(12a)- H(12f5CHo) is clearly evident corresponding to a twenty-fold 13 enhancement of the

C-12a-methyl signal which also is an internal

check for the enrichment achieved.

These results (20) impose strict

requirements on the mechanism of corrin synthesis with respect to retention of the C-l methyl protons and were confirmed by independent methods at Zurich (21) and Cambridge (22).

Thus, unless an exchanged proton from C-l

CHg is returned regio- and presumably stereo-specifically, structures involving an exo-methylene at C-l can be eliminated as part of the biosynthetic assembly of corrin from seco-corrin as envisaged in the schemes developed at the end of this discussion. C

00

05

PPM FIGURE 2

10

12/3

^ 2a,12a,17)8

15

25U

D.

Formation of Uro'gen I I I and i t s Role in Corrin Biosynthesis

Much of our earlier work at Yale on the uro'gen I I I - c o r r i n connection has recently been summarized (23) and only the salient features necessary for the ensuing discussion will be mentioned here.

In 1971-1972 the only

references (24,25) to uro'gen I I I (4) as an obligatory precursor of vitamin B-J2 w e r e extremely discouraging. However, with the establishment of proper pH control and feeding conditions, we found reproducible conversion of 13 [8- C]-PBG and of the chemically synthesized type I - I V uro'gen mixture labeled in the propionate side chains to specimens of dicyanocobalamin 13 whose C-NMR enrichments [four carbons in each case at 25.9, 26.8, 27.8 and 34.1 ppm] occurred with identical chemical s h i f t (Figure 1, Table 1). Since uro'gen I (5j had been shown to be quite ineffective in labeling B-^, the conclusion seemed inescapable — u r o ' g e n I I I must be the precursor of vitamin B 1 9 (6). This finding restored considerable confidence and led to confirmation by a further set of 13 C labels inserted into uro gen I I I by •

•

total regiospecific synthesis at C-5 and C-15 (Yale) and of

14

•

C at CHgCC^H

in ring C (Cambridge), and the appearance of enrichments at C-5, C-15 (108.4 and 105.2 ppm) in the cyanocobalamin derived from whole cell feeding of these precursors and of radioactivity at C-12B (•) in cobester as shown in Figure 3 (26), results which were also confirmed at Stuttgart (15).

-COR, '•^COR,

R, = NH 2 • '«C

R 2 = IPA-Phosphate-Ribose-DMBI—Co FIGURE 3

255 A parallel series of experiments with [5,15- 1 4 C 2 ]- and [5,15,20- 1 4 C 3 ]uro'gen disclosed that, after proper care was taken to solve the severe problems associated with the chemical production of formaldehyde from the meso positions of uro'gen, a stoichiometric amount of formaldehyde from C-20 of uro'gen could be trapped (as the dimedone adduct) during the conversion of uro'gen III to cobyrinic acid in the cell-free system described above (27).

The logical but unlikely possibility exists in this experiment

that, by an amazing coincidence, the radioactive yield ( - 3 % ) of cobyrinate from

14

C at C-5 and C-15 of uro'gen (analyzed as cobester) fortuitously 14 matches that of formaldehyde from C-20! We recall at this juncture Shemin's classic experiment (4) with [5-^C]-ALA in which ca. 10% of the radioactivity from the labeled position must have been returned to the "C-l" pool in order to explain the appearance of a small amount of radioactivity in the methyl groups of B-|2> including C-l.

We shall return to

the logic of this fascinating point during the discussion of the structures of the methylated isobacteriochlorins. In this review, we have reached 1976-1977 which coincides with the availability of low and high resolution microprobe NMR experiments and new facilities in Texas which allowed us to initiate a study of the application of low temperature, high resolution CMR spectroscopy to the enzymes of the B-|2 pathway.

As a first example we chose the deaminase/cosynthetase-

mediated conversion of PBG to uro'gens I and III.

This work sets the stage

for subsequent experiments designed to trace the complete metabolism of glycine to corrin via cryo-enzymological

NMR techniques.

At the same time

we have studied metabolism in the intact, living cells of £_. shermanii in the NMR tube, and will refer to this technique at the end of our discussion. The formation of uro'gen III requires the participation of two enzymes, PBG deaminase (uro'gen I synthetase) and uro'gen III cosynthetase (28). In the presence of deaminase alone, PBG is converted into uro'gen I (5J which is not further involved in the pathway of tetrapyrrole or corrin biosynthesis.

We have followed the events of enzymic conversion of PBG (2), 13

enriched with

C, into uro'gens in the NMR tube, thus avoiding the isola-

tion of potentially labile compounds. In a typical experiment, the highly purified deaminase (80 units per mg) was incubated with 300 yg of [11- 13 C]-

256 PBG (Figure 4) in tris buffer at 37°C under anaerobic conditions.

NMR

spectra were recorded on a Varian SC-300 NMR spectrometer using single frequency off-resonance proton decoupling.

(A)

(P)

FIGURE 4

The spectrum of uro'gen I, obtained after the enzyme reaction had been allowed to proceed to completion, is shown in Figure 5a. centered at 21.63 ppm-(reduced V ^ ^ H

The triplet

= 100 Hz) results from the four

equivalent meso carbon atoms (5, 10, 15 and 20 in uro'gen I).

When the

enzyme reaction is allowed to proceed until 47% of the PBG has been consumed (11 min), in addition to the remaining PBG signal (triplet centered at 34.95 ppm, reduced

1

J 1 3 C - 1 H = 125 Hz) and the uro'gen signal

(21.63ppm)

at 0°C, a complex signal is also present at 21.85-22.15 ppm which inte13 grated as 17% of the C (Figure 5b). In addition, a further signal 13 (integral: 5% of C) appears as a triplet at 54.78 ppm (reduced 113 1 J

C- H = 130 Hz).

These latter signals disappear as the enzymic con-

version proceeds to completion.

Treatment of the incubation medium with

either acid or base under conditions where the deaminase is inactive affords only uro'gen I which is also the sole product of the reaction with deaminase in buffer.

When the NMR experiment is performed under

identical conditions in the presence of sufficient cosynthetase (29) to produce 100% of uro'gen III, after 47% of the PBG has been consumed

257

(11 min), none of the complex signals at 21.85-22.15 ppm or at 54.78 ppm is observed, whilst the uro'gen (III) peak is proportionally increased (Figure 5c).

(a)

FIGURE 5.

75.5 MHz C spectra of enriched uro'gen I (a), uro'gen I and pre-uro'gen (b), and uro'gen III (c) at 0°C under single frequency off-resonance proton decoupling conditions. Typically, 35,000 90° pulses were accumulated over a spectral width of • 9,000 Hz while locked to internal D 2 0 (10%) with a repetition rate of 0.6 seconds. The lines were broadened approximately 2 Hz by exponential multiplication of the F.I.D. (30).

258

From these data we conclude that during the enzymic conversion of PBG into uro'gen a hitherto undetected intermediate is accumulated in solution which 13 ultimately yields uro gen I. The absence of C signals at 21.85-22.15 and 54.78 ppm under conditions where the enzymic system is forming only uro'gen III suggested that the intermediate, termed pre-uro'gen, responsible for these signals may be involved in the normal pathway of uro'gen III biosynthesis, thus favoring the hypothesis of rearrangement at the tetrapyrrole level and the formation of pre-uro'gen from an enzyme-bound version of the bilane (6j.

The chemical shifts of pre-uro'gen correspond to one of

the intermediates on the energy surface represented by the uro'gen I tautomer (7J, the N-alkylated macrocycle (8j and the spiro compound (9j. The appearance of signals at 54.78 ppm (integral 1C) and at 21.85-22.15 (3C) at present favor (8) as the structure of pre-uro'gen, since acid or base treatment affords only uro'gen I, whereas the action of cosynthetase produces exclusively uro'gen III. bilane (6) [no

13

The chemical shift data exclude the

CH 2 NH 2 at 34.64 ppm (30)] but still leave the spiro

compound (9), the uro'gen I tautomer (7J and the modified bilane (6) Structure (6) is further

( C ^ N ^ = CH,,0H) as formal possibilities.

excluded on the basis of the ratio of uro'gens reported for the nonenzymic cyclization of this species (31), although type III uro'gen formation has been demonstrated during the combined action of high concentrations of deaminase and cosynthetase on (6). 13 These alternatives were further narrowed when [2,11-

C2]-PBG served as

substrate (see Figure 6). In this experiment the resultant signal at 1 lo 1 54.78 ppm appeared as a broad triplet (reduced J C- H = 130 Hz), thus 13» 13« excluding (7J which would exhibit C- C coupling for this carbon. The inherent instability of pre-uro'gen ( t ^ allow a final decision between 8 and

=

4 min at 37°C) so far does not

since these are formally connected

by allowed [1,5]-sigmatropic shifts with the type I and type III uro'gens (e.g., 8

-*• £ and 9 t 8

7).

There is, however, no doubt that the

function of synthetase is much more complex than had previously been thought since it is capable of fashioning a highly reactive intermediate (e.g., 8^ and/or 9J which is then either rearranged chemically to uro'gen I or, in the presence of cosynthetase, is diverted to uro'gen III.

259

• From C - 2 of PBG

• From C-11 of PBG FIGURE 6

It now appears that the exact nature of the pathway from PBG to uro'gen III and, in principle, from uro'gen III to corrin can be determined by further application of this methodology, which has the advantage of using only the natural substrate, PBG.

In our view, the earlier and sometimes conflicting

results with pyrromethanes obtained in this laboratory and elsewhere (28) can be ascribed in part to chemical interaction of these molecules with pre-uro'gen and subsequent formation of uro'gen III.

Further biochemical

evidence that pre-uro'gen is indeed the long-sought substrate for cosynthetase was obtained as follows.

The deaminase-free pre-uro'gen filtrates

(stage 1) were incubated with uro'gen III cosynthetase (32) (16 u/ml, 37 u/mg)* together with the appropriate controls (stage 2) which showed the complete absence of deaminase, since during stage 2 there was no further consumption of PBG.

The amounts of uro'gen III formed during

these experiments are shown in Table 2.

*

One unit of uro'gen III cosynthetase is equivalent to 1 ymol pre-uro'gen/hr determined under standard uro'gen III cosynthetase assay conditions.

260 TABLE 2

Incubation Time (mins)

% Isomer III Formed

Stage 1

Stage 2

deaminase

cosynthetase

11

a

cosynthetase

10

% of Pre-uro'gen Present

% Conversion of Pre-uro'gen into Uro'gen III

buffer

37

a

0a

43 c

86

21

a

a

c

87

23

10

37

10

7a

0a

0

60

10

0a

0a

30

10

13 b

0b

24 -

0C -

Determined by hplc of uroporphyrin methyl esters.

^Determined by hplc of coproporphyrin methyl esters. 13 c Determined by C NMR in a separate experiment.

The data clearly demonstrate several important features of the uro'gen III synthesizing system,

a)

Pre-uro'gen is formed transiently by the action

of PBG deaminase and, in the absence of uro'gen III cosynthetase, rearranges to form uro'gen I in acidic, basic or neutral media,

b)

The

formation of uro'gen I from pre-uro'gen occurs in the absence of deaminase, c)

Pre-uro'gen is converted into uro'gen III in high yield by uro'gen III

cosynthetase.

d)

PBG deaminase is not required for the conversion of

pre-uro'gen into uro'gen III.

e)

Pre-uro'gen is tetrapyrrolic since PBG

is not consumed during its conversion to uro'gen III.

f)

Uro'gen I does

not act as a substrate for uro'gen III cosynthetase. All of the evidence suggests that a hitherto unsuspected pathway for the biosynthesis of uro'gen III is operative in which the enzymes act sequentially and independently as shown below, with pre-uro'gen as the key intermediate (32). . ^ e o v ^ porphobilinogen

uro' gen I

deaminase. uro'gen III

261 The properties of pre-uro'gen require that the rearrangement takes place after head-to-tail assembly (28) and not, as previously suggested, at an earlier stage (28a,32). The discovery of pre-uro'gen is not only a splendid example of detection of a labile intermediate in the corrin pathway by 13 CMR spectroscopy but also explains the synergistic relationship of deaminase and cosynthetase, and reconciles previously conflicting viewpoints concerning the timing of the switch mechanism in the type I I I problem.

Thus, regardless of the mechanism whereby dipyrromethanes (as 1_0)

are inserted intact* into the biosynthetic machinery, the evidence now favors an enzyme-bound version of transformation to pre-uro'gen.

as the most l i k e l y species undergoing

I t has very recently been established (34)

that the CMR spectrum of the bilane (6) obtained by total synthesis does not correspond with that of pre-uro'gen, 13that (§}c i s not^ a substrate for purified cosynthetase and, by using [11- C, 1- N]-enriched PBG as substrate, that pre-uro'gen indeed has the unique structure 8 which displays one bond

E.

13

C - 1 5 N coupling in both the

13

C and

15

N NMR spectra.

The Final Stages. Characterization and Intermediacy of the Isobacteriochlorins of P_. shermanii

As soon as uro'gen I I I was defined as the precursor of the corrin nucleus (Figure 3) the search for p a r t i a l l y methylated intermediates on the way to B-|2 began in earnest and i t was noted as early as 1973 (35) that the preferred structure for sirohydrochlorin, the iron-free prosthetic group of the enzyme siroheme, could be modified to accommodate i t s possible role as a biosynthetic intermediate.

At f i r s t sight this would have seemed an

extraordinary coincidence but as the study of sirohydrochlorin developed

*

A recent suggestion (28a) that the DPM (1_0) suffers fragmentation to a monopyrrole and reassembly to bilane cannot be correct since uro'gen I is not labeled in this experiment.

(XM P A

A P

H

H

10

262

13A TABLE 3.

Proton chemical shifts and assignments of sirohydrochlorin methyl ester from F\ shermanii cell-free homogenate (A) and from £. coli NADPH-sulfite reductase (B) a

Position b

A

B

15

8.536

8.537

1H, s

10 or 20

7.461

7.464

1H, s

10 or 20

7.361

7.359

1H, S

5

6.777

6.778

1H, S

18a, 12a

4.284

4.286

4H, S

3, 8

4.1

4.1

2H, M

13a, 17a

3.77

3.77

1

-CO2CH3

3.722, 3.716, 3.,671 3.667, 3.651 , 3..612 3.592

3..722, 3.717, 3..670 3.,668, 3.653, 3..612 3.,591

13b, 17b

2.968, 2.938, 2..910

2.,964, 2.940, 2..907

4H, t

2a, 7a

2.750, 2.734, 2..724

2..752, 2.733, 2..725

4H, M

3b or 8b

2.610, 2.583, 2..555

2..609, 2.580, 2,.552

2H, t

2.0-2.4

2.0-2.4

8H, M

3a, 8a, NH and 3b or 8b 2, 7 (CH 3 ) a

1.869, 1.828

1.,869, 1.828

The chemical shifts are quoted in ppm downfield from TMS. were obtained at 270 MHz in C D C U . b See structure 13A above for the numbering system.

I (

28H

3H, s The spectra

263 in parallel with the isolation of orange-fluorescent substances from cobalt-deficient, ALA-supplemented incubations of P_. shermanii, complete identity of the dimethyl isobacteriochlorin from the 6-electron reducing enzyme and from the B-^-producing organism was established.

Thus, inspec-

tion of the UV, CD, Mass and PMR (Table 3) spectral data for the methyl ester of the P. shermanii metabolite and of sirohydrochlorin from £. coli sulfite reductase leaves no doubt that the substances, which also show complete correspondence in TLC R^ values, are identical in every respect. The molecular constitution was also confirmed by high resolution mass determination of the molecular ion (974.4167) which revealed the composition C 5 0 H 6 2 N 4 0 1 6

(974.4160).

The UV spectrum of sirohydrochlorin is diagnostic of the isobacteriochlorin class requiring the two reduced rings to be adjacent.

Since sirohydro-

chlorin is incorporated intact into cobyrinic acid (see later), structures 11-14

represent the possible alternatives.

The PMR assignments given in Table 3 were made as follows.

The 3H singlet

resonances at 6 1.87 and 1.83 are consistent with the known chemical shifts of methyl groups on the reduced ring of a chlorin.

Similarly the 2H

multiplet at 6 4.1 also falls within the range of shifts expected for methine hydrogens on such rings.

The literature model system that is most

relevant to the remaining assignments is a-tetrahydrooctaethylporphyrin, which is an isobacteriochlorin.

Application of the chemical shift differ-

ences between the latter and octaethylporphyrin allowed the assignments given in Table 3 (see formula 13A for the numbering system). As required by this assignment, irradiation of the partially hidden multiplet at 6 3.77 (C-12a, 17a) resulted in the 6 2.94 triplet (C-13b, 17b) collapsing to a singlet.

Additionally, irradiation at frequencies between

6 2.0-2.4 simplified the 6 4.1 multiplet and vice versa, thus confirming that the methine hydrogens are coupled to the methylene hydrogens (C-3a, 8a) of the reduced-ring propionate groups.

The 2H triplet 6 2.50 was found to

be coupled to the 6 2.0-2.4 multiplet.

It is, therefore, probably one of

the methylene groups at C-3b and C-8b which has been shifted downfield from the other by a subtle steric or electronic effect.

The 8H multiplet

261+

at S 2.0-2.4 i s assigned to the two NH resonances and the remaining reduced-ring propionate methylene hydrogens (C-3b, 8b).

Such a chemical

s h i f t would not be unusual since the NH resonances o f o c t a e t h y l i s o b a c t e r i o c h l o r i n were reported as 6 2.96 (CDClg) and 1.77 (CgDg). The 6 2.72-2.75 multiplet (C-2a, 7a) i s not coupled to any other resonances. An a l t e r n a t i v e structure to (1_3) having r i n g D methylated would have structure (lj4).

However, the l a t t e r would require coupling between the

reduced-ring acetate methylene hydrogens (6 2.72-2.75 and the neighboring methine hydrogen (6 4 . 1 ) .

At t h i s stage in the i n v e s t i g a t i o n the structural

proposals f o r s i r o h y d r o c h l o r i n were considerably reduced by biosynthetic experiments.

To c i t e one of several examples, a doubly-labeled sample of

reduced s i r o h y d r o c h l o r i n methyl ester (1_5, OH = OCHo) was prepared by 14 3 separate incubations with [4- C]-ALA and [ H,C]-S-adenosylmethionine. 3 14 When the samples were combined ( H/ C = 3.45), p u r i f i e d and re-fed a f t e r h y d r o l y s i s and sodium amalgam reduction (1_5), the r e s u l t a n t cobester (3^; R = Me) was obtained in 2.4% radiochemical y i e l d with retention of the r a t i o ( 3 H/ 1 4 C = 3.41) (see Figure 7). In view of the correspondence of the oxidation level of 1_5 and o f i t s b i o synthetic product, cobyrinic acid (3^; R = H), i t was s u r p r i s i n g to f i n d that incubation of the unreduced species 13 led to equally good (0.3-2.8%) incorporation.

We d i s c u s s below the implication of t h i s f i n d i n g .

At t h i s

stage in the i n v e s t i g a t i o n Mu'ller (36) described the i s o l a t i o n and c e l l free conversion of a

shermanii metabolite, Faktor I I , to c o b y r i n i c acid.

Comparison o f the reported physical data for Faktor I I and s i r o h y d r o c h l o r i n leaves l i t t l e doubt that they are i d e n t i c a l . Two s t r u c t u r e s , (12J and ( 1 3 ) , are s t i l l

consistent with the spectroscopic

and bioincorporation data mentioned above.

However, since i t i s

unlikely

that methylation of r i n g C occurs before decarboxylation of the acetate side chain during B ^ b i o s y n t h e s i s , structure (13) becomes a t t r a c t i v e for sirohydrochlorin.

This complete stereostructure 1_3 was confirmed by a 13 s e r i e s of biosynthetic experiments in which C - l a b e l i n g again proved to be of diagnostic value in a r r i v i n g at a unique s o l u t i o n .

I t was f i r s t

shown that a natural abundance CMR spectrum could be obtained on a 500 yg

265

V f

COOH

COOH

COOH COOH (13)

P

H P H

p

COOH-

/»,.«Cell-Free

A

v y ^ ^ ^ L . P

COOH

System COOR-^I

\\

*

COOR

AOOR

COO

COOI

COOR A=3H »=mC

(3)

^ « 3.45

"COOR R=MeJ=3.4l

FIGURE 7

sample in which 10 13 tion of [4-

transients were collected over 252 hrs.

Next, incuba-

C]-ALA (90% enriched) with the P. shermanii cell-free homoge-

nate yielded a purified sample of sirohydrochlorin (400 yg) whose protondecoupled and proton-coupled spectra showed that only structures 12_ and are3 compatible with the observation of a doublet (J = 135 Hz) for each sp carbon resonance (C-3 and C-8), since structures 11_ and 14 would 3 exhibit only one such enriched sp carbon-bearing hydrogen. 13 Finally, incubation with [5chlorin has structure 1_3. Figure 8.

C]-ALA enabled us to show that sirohydro-

The expected labeling pattern is as shown in

In the proton decoupled spectrum (Figure 8, spectrum A), the

C-15 resonance is a triplet (J = 71.1 Hz) due to 1,2 coupling with two adjacent enriched sites.

(A lower intensity doublet is also present due

266

f

J

(4,I6),20=6Hz

H

A

A-

C-20

H

C-IO J

9,I0

=

77.5 H z

J|0|14 = 4.4Hz

r S P

H

C-15

J|5,(I4,I6) = 71.1Hz

C-5

rr

i S

J

4 , 5 » 71.0 Hz

J 5 f 9 » 5.7 H z

100

90

PPM FIGURE 8

P

267 to those molecules having only one adjacent enriched site.)

The C-5 and

C-10 resonances both occur as doublet of doublets due to 1,2 and 1,4 couplings whilst C-20 shows only 1,4 interactions.

The four meso-hydrogen

PMR resonances occurred as doublets at 6 8.54, 7.46, 7.36 and 6.78 ppm. As discussed by Bonnett et^ al_. (37), the upfield meso-hydrogen resonance of an isobacteriochlorin may be assigned to that between the reduced (methylated) rings and the downfield resonance to that between the nonreduced rings.

Thus, the 6 8.54 hydrogen would be coupled to C-15 in

structure 1_3 and to C-20 in structure 12^.

The former case was confirmed

by selective irradiation of the 6 8.54 hydrogen resonance.

As can be seen

from spectrum (B) in Figure 8, the C-15 resonance at 6 107.4 remained a triplet whilst the other meso-carbon resonances showed coupling to mesohydrogen resonances. The other meso-carbon resonances were assigned as follows.

During selec-

tive irradiation of the upfield meso-hydrogen resonance at 6 6.78 (Figure 8C) the upfield meso-carbon resonance at C-10 ~ C-20 > C-5) parallel that of the meso-hydrogen chemical shifts, being a reflection of the relative charge density at each meso-carbon.

All of these spectra

were determined on the octamethyl ester of ]_3. Our colleagues in Stuttgart, Cambridge and Moscow have recently isolated metabolites from P^. shermanii with similar UV and mass spectral characteristics to sirohydrochlorin and their work is described in Chapter 2 of this volume.

The Anglo-Russian group postulated structure 13^ for

their metabolite after assuming that it was on the B ^ pathway and further that ring C was not methylated.

Further work with the lactones and free

esters of this series (38) independently confirmed the above conclusions and showed that sirohydrochlorin is indeed identical with a P. shermanii metabolite, that it has structure corrin pathway.

and that it is an intermediate on the

The possibility that siroheme represents a prebiotic

268

sulfate-reducing agent (35) and further, that both sirohydrochlorin and vitamin B-|2~producing anerobic organisms predate the evolution of hemesynthesizing aerobes (23), suggests that the reductive methylation of reduced porphyrins may be a phenomenon of considerable antiquity (three billion years).

We also note that the same dimethylisobacteriochlorin

prosthetic group is active in the enzymes of nitrite (39).

ammonia reduction

Thus, it appears that the biosynthetic architecture of corrins

evolved at a very primitive stage of life on this planet and that the problem we are investigating has existed for even longer than we had realized at the outset.

The intermediacy of the dimethylisobacteriochlorin

(13) (40) in corrin biosynthesis requires not only reappraisal of the specific incorporation of uro'gen III heptacarboxylic acid into cobyrinic acid, which could be explained by non-specific enzymatic conversion of a substrate closely related to, but not identical with, the normal metabolic intermediate, but also a significant modification of our present working hypothesis (41) for the post-uro'gen III segment of corrin biosynthesis. With the knowledge that 1_3 is established as an intermediate by isolation in cobalt-deficient incubations and by intact specific incorporations into cobyrinic acid (thereby defining the absolute stereochemistry of sirohydrochlorin), the remaining stages of the sirohydrochlorin-cobyrinic acid pathway appear, not necessarily in this order, to involve:

a) introduction of

five additional methyl groups from S-adenosylmethionine with retention of the methyl protons;

b) loss of C-20;

system;

c) reclosure of the seco-corrin

d) 2-electron reduction to corrin; 2+ 3+ valency change Co Co .

e) insertion of cobalt with

As a guide to future experimentation we recently offered (40) a speculative mechanistic rationale of these processes based on the known or presumed chemistry of the reduced porphyrins and of corrins.

Many of the ideas

embodied in the Schemes are presently being tested experimentally and include certain key modifications of a proposal for corrin biosynthesis published several years ago (41), some of which stem from the recent elegant chemical analogies uncovered in Eschenmoser's (42) several approaches to corrins.

synthetic

These Schemes also embodied the observation that

two new metabolites (Faktors I and III) had been isolated at Stuttgart (36, 43) and that Faktor III appeared to be related to the bislactone corriphyrin-3 (44).

269

SAM

P FAKTOR I

P REDUCED

SIROHYDROCHLORIN (=FAKTOR I) REDUCED

P

p

SIROHYDROCHLORIN (FAKTOR I ) SCHEME 1

Scheme 1 shows the incorporation of sirohydrochlorin (= Faktor I I ) and the presumably intact incorporations of Faktors I and I I I all taking place at the reduced level.

The experimental observation of incorporation of

Faktors I I and I I I (43), but only the reduced form of Faktor I (36) and uro'gen I I I , indicates the presence of a non-specific oxido-reductase system which, however, is not capable of reducing either uro I I I to uro'gen I I I or Faktor I.

The evidence for the structures suggested for Faktors I

and I I I are permissive rather than diagnostic. methylated in ring B rather than in ring A.

Thus, Faktor I could be

Faktor I I I , while undoubtedly

a trimethyl isobacteriochlorin to which the structure (16) has been assigned (44) on NMR evidence, could, in fact, be another isomer and in 14 the absence of location of C label, does not yet occupy the position of proven intermediacy in cobyrinate biosynthesis.

270

P

P 16

P

P

1_7 A = CH 2 C0 2 H P = CH 2 CH 2 CO 2 H 18

A = CH 2 C0 2 CH 3 P = CH,CH O C0 O CH

In a recent collaborative study with Professor G. Mliller we have provided spectroscopic and biochemical evidence that the "extra" methyl group is added to sirohydrochlorin at C-20 rather than at C-5 leading to the revised structure (17J for Faktor III, i.e. 20-methyl sirohydrochlorin, which as described by Professor Miiller (see Chapter 2 ) suffers loss of both C-20 and its attached methyl group during biotransformation to cobyrinic acid. Faktor III was isolated from S-aminolevulinic acid (ALA)-supplemented cobalt-free incubations of P^. shermanii (ATCC 9614) and from a B ^ deficient mutant (45) of this organism.

High resolution FD mass spec-

trometry of the octamethyl ester (18) established the formula C5i H 64 N 4^16 (988.4342) and analysis of the PMR spectrum (300 MHz) revealed only three signals at 6 6.43, 7.21 and 8.33 ppm in contrast to the four signals in this region in the spectrum of (13) which have been assigned to the four meso protons at C-5, C-10/C-20 and C-15 (6 6.78, 7.36/7.46 and 8.54, respectively).

Faktor III is therefore 10- or 20-methylsirohydrochlorin.

In order to decide between these alternatives a specimen of Faktor III (400 yg) was prepared from a suspended-cell incubation from shermanii 13 13 13 CH-j-methionine and [5- C]-ALA. When this C-enriched

containing

species (as the octamethyl ester) was examined by microprobe CMR spectroscopy (Figure 9) it became possible to deduce the complete structure (18).

First, the downfield position of the C-15 meso-carbon triplet at

272 6 108.98 (J = 72 Hz) confirms that rings A and B are methylated (40), and 13 13 since the meso-carbon signals at 6 89.5 and 95.4 each show C- Ccoupling to an enriched neighbor (J = 70 Hz) these are assigned to C-5 and C-10, respectively, by analogy with the corresponding resonances in sirohydro13 chlorin derived by biochemical enrichment with [5- C]-ALA (40). Thus, the remaining meso-carbon resonance at 6 104.8 which consists of a doublet (J = 44.5 Hz) must correspond to C-20, the additional fine being 13 structure 13 That the C- C coupling

due to long-range coupling with C-4 and C-16.

constant of 44.5 Hz for C-20 is due to substitution by a methionine-derived methyl group is confirmed by inspection of the methyl region of the CMR spectrum which displays three enriched species consisting of singlets at 6 20.17 and 19.62 and a doublet at 6 18.79 (J = 44.5 Hz).13 It can be seen that, due to different efficiencies of incorporation of C-SAM and of 13 [5- C]-ALA, the enrichments in the methyl groups and in the ALA-derived 2 sp

carbons are not identical.

Hence the satellite intensities reflect a

greater enrichment in C-20 than in its pendant methyl group and allow unambiguous assignment of structure

to the octamethyl ester.

Thus

Faktor III is 1_7, i.e. 20-methylsirohydrochlorin, rather than the C-5 methylated isobacteriochlorin, contrary to what had previously been thought (44).

The absolute stereochemistry of Faktor III and its relationship to

cobyrinic acid was obtained by the following biochemical experiment. 3

described by Professor Miiller (Chapter 2 ), the double-labeled ( H /

14

As C)

20-methylsirohydrochlorin is converted to cobyrinic acid with loss of the "C2" unit consisting of C-20 (derived from C-5 of ALA) and methyl group originating from methionine.

An alternative explanation, that the C-20 3

methyl group migrates to C-l (or -> C-l9 -»• C-17) with complete loss of

H

can be ruled out, for earlier experiments have shown that all seven methionine-deri ved methyl groups of B ^

are inserted without exchange of

the CH 3 protons (20,21 ,22). The nature of the loss of C-20 (from C-5 of ALA) in corrin biosynthesis must now be explained.

In earlier experiments it was clearly shown (26, 14 27) that, under carefully controlled conditions, C formaldehyde could be trapped from the C-20 position of uro'gen III. above can be interpreted in several ways:

The data presented

a) methylation at C-20 is

followed by loss of a "C ? " unit which is further cleaved to "C-," units,

273 one of which is trapped as formaldehyde;

b) the formaldehyde is released

[under enzymic control (27)] only from the uro'gen III molecule and not from Faktor III which could release a "C2" unit;

c) more than one pathway

exists for the biotransformation of uro'gen III to cobyrinic acid. appears that in order to achieve the inter-corrin A

It thus

D ring junction the

biosynthetic route requires not only the specific formation and subsequent disruption of the type III uro'gen macrocycle but the sacrifice of at least one methionine-derived methyl group and the carbon to which i t is attached (C-20), since both of these must be excised from the species undergoing (or having undergone) seco-corrin

corrin (41,46) closure.

This apparently

prodigal series of events is portrayed in Scheme 2 which represents only one of several working hypotheses consonant with published data.

Further

work is in progress to c l a r i f y post-Faktor III metabolism in vitamin B^ biosynthesis.

FAKTOR ID REDUCED

SCHEME 2

21k

As portrayed in Scheme 2, decarboxylation at ring C, methylation (SAM) and ring opening can lead via loss of C-20 either directly to dehydrocorrin (41,46) or, in an interesting variant, by methylation at C-l to the 1,19 dimethyl seco-corrin whose electrocyclic closure reveals a 1,19 transmethyl dehydrocorrin.

The last part of the sequence (Scheme 3) portrays

the 1,5 shifts of the B-oriented C-19 methyl in ring D to C-17, its final resting place on the upper face of the molecule.

A splendid analogy for

this step has been discovered by A. W. Johnson (47).

The pathway could

then terminate by two-electron reduction to cobalt-free cobyrinic acid followed by, or synchronized with, insertion of cobalt (41).

It is hoped

that,by examining intact cells and enzymes by NMR (wide bore-high resolution),corrin biosynthesis can be "viewed" by a non-invasive technique already of great promise at low resolution (48).

SCHEME 3

275 Obviously, the next few years will provide many formidable problems (and even more surprises) in uncovering the final details of corrin biosynthesis. While eagerly anticipating the continuing challenge in the dynamics of molecular architecture, it is appropriate at this stage to pay tribute to the outstanding energy and devotion of my younger colleagues who have contributed so much to the progress of the work described in this lecture and whose names are mentioned in the references.

The microbiological and

analytical techniques necessary for this exacting work with sub-milligram quantities of enriched, purified isolates were superbly handled by A. Brown, D. Brownstein, D. Stem, J. Petrillo, P. Copsey and M. Smith.

Our

work at Yale and Texas A&M has been made possible by grants from the National Institutes of Health (AM 20528), the National Science Foundation (CHE 77-04877) and The Robert A. Welch Foundation (A-713).

References 1.

Bray, R., Shemin, D.:

2.

Vitamin B-j2 and Intrinsic Factor, 2nd European Symposium, Hamburg (1961), Ferdinand Erke, Verlag, Stuttgart, 1962.

3.

Bernhauer, K., Wagner, F., Michna, H., Rapp, P., Vogelmann, H.: Hoppe-Seyler's Z. Physiol. Chem. 349, 1297 (1968).

4.

Shemin, D., Bray, R. C.: Bray, R. C., Shemin, D.:

5.

Mathewson, J. H., Corwin, A. H.:

6.

(a) Scott, A. I., Townsend, C. A., Okada, K., Kajiwara, M., Whitman, P. J., Cushley, R. J.: J. Am. Chem. Soc. 94, 8267 (1972); (b) Scott, A. I., Townsend, C. A., Okada, K., Kajiwara, M., Cushley, R. J.: J. Am. Chem. Soc. 94, 8269 (1972); (c) Scott, A. I., Townsend, C. A., Okada, K., Kajiwara, M., Cushley, R. J., Whitman, P. J.: J. Am. Chem. Soc. 96, 8069 (1974).

7.

Brown, C. E., Katz, J. J., Shemin, D.: 68, 1083 (1971 ).

8.

Battersby, A. R.,Ihara, M., McDonald, E., Stephenson, J. R.: Soc., Chem. Commun., 404 (1973).

9.

Bonnett, R., Godfrey, J. M., Math, V. B., Scopes, P. M., Thomas, R. N.: J. Chem. Soc., Perkin Trans. 1 , 252 (1973); Bonnett, R., Godfrey, J. M., Math, V. B., ibid. (C), 3736 (1971 ).

10.

Biochim. biophysica acta 30, 647 (1958).

Ann. N.Y. Acad. Sei. 11^, 615 (1964); J. Biol. Chem. 238, 1501 (1963). J. Am. Chem. Soc. 83, 135 (1961).

Proc. Natl. Acad. Sei.(U.S.A.)

Stoeckli-Evans, H., Edmond, E., Hodgkin, D. Crowfoot: Perkin Trans II, 605 (1972).

J. Chem.

J. Chem. Soc.,

276 11.

Scott, A. I., Towrisend, C. A., Cushley, R. J.: 5759 (1973).

J. Am. Chem. Soc. 95,

12.

Doddrel1, D., Allerhand, A.: (1971).

13.

Battersby, A. R., Ihara, M., McDonald, E., Stephenson, J. R., Golding, B. T.: J. Chem. Soc., Chem. Commun., 458 (1974).

14.

Scott, A. I., Yagen, B., Lee, E.:

15.

Battersby, A. R., Ihara, M., McDonald, E., Satoh, F., Williams, D. C.: J. Chem. Soc., Chem. Commun., 436 (1975); see also Dauner, H.-0., Müller, G.: Hoppe-Seyler's Z. Physiol. Chem. 356, 1353 (1975).

16.

Dauner, H., Müller, G.: (1975).

17.

Lee, E., Yagen, B.: Unpublished work, Yale University, 1973; Lee, E.: Ph.D. Thesis, Yale University, 1974.

18.

Brown, C. E., Shemin, D., Katz, J. J.:

19.

Hensens, 0. D., Hill, H. A. 0., Thornton, J., Turner, A. M., Williams, R. J. P.: Philos. Trans. R. Soc. London, Ser. B, 273, 353 (1976).

20.

Scott, A. I., Kajiwara, M., Takahashi, T., Armitage, I. M., Demou, P., Petrocine, D.: J. Chem. Soc., Chem. Commun., 544 (1976).

21.

Imfeld, M., Townsend, C. A., Arigoni, D.: 541 (1976).

22.

Battersby, A. R., Hollenstein, R., McDonald, E., Williams, D. C. : J. Chem. Soc., Chem. Commun., 543 (1976).

23.

Scott, A. I.:

24.

Müller, G., Dieterle, W. : (1971).

25.

Franck, B., Gatitz, D., Hüper, F.: 421 (1972).

26.

Scott, A. I., Yagen, B., Georgopapadakou, N., Ho, K. S., Klioze, S., Lee, E., Lee, S. L., Temme, G. H., Townsend, C. A., Armitage, I. M. : J. Am. Chem. Soc. 97, 2548 (1975).

27.

Kajiwara, M., Ho, K. S., Klein, H., Scott, A. I., Gossauer, A., Engel, J., Neumann, E., Zilch, H.: Bioorg. Chem. 6, 397 (1977).

28.

Reviewed by (a) Battersby, A. R., McDonald, E. : Acc. Chem. Res. 1_2, 14 (1979); (b) Frydman, B., Frydman, R. B.: ibid. 8, 201 (1975).

29.

Jordan, P. M., Nordlöv, H., Schneider, M. M., Hosozawa, S., Scott, A. I.: submitted for publication.

30.

Burton, G., Fagerness, P. E., Hosozawa, S., Jordan, P. M., Scott, A. I.: J. Chem. Soc., Chem. Commun. (1979) in press.

31.

Battersby, A. R., Fookes, C. J. R., McDonald, E., Meegan, M. J. : J. Chem. Soc., Chem. Commun., 185 (1978).

32.

Jordan, P. M., Burton, G., Nordlöv, H., Pryde, L. M., Schneider,M. M., Scott, A. I.: J. Chem. Soc., Chem. Commun. (1979) in press.

Proc. Natl. Acad. Sei.(U.S.A.) 68, 1083

J. Am. Chem. Soc. 95, 5761 (1973).

Hoppe-Seyler's Z. Phsiol. Chem. 356, 1353

Acc. Chem. Res. H ,

J. Biol. Chem. 248, 8015 (1973).

J. Chem. Soc., Chem. Commun.,

29 (1978).

Hoppe-Seyler's Z. Physiol. Chem. 352, 143 Angew Chem., Int. Ed. Engl. 11_,

277

33.

Scott, A. I., Ho, K. S., Kajiwara, M., Takahashi, T.: Soc. 98, 1589 (1976).

J. Am. Chem.

34.

Scott, A. I., Burton, G., Nordlöv, H., Fagerness, P. E., Matsumoto, H., Hosozawa, S.: J. Am. Chem. Soc. (1979) in press.

35.

Siegel, L. M., Murphy, M. J., Kamin, H.: J. Biol. Chem. 248, 251 (1973); Murphy, M. J., Siegel, L. M., Kamin, H., Rosenthal, D.: ibid. 248, 2801 (1973).

36.

Deeg, R., Kriemler, H.-P., Bergmann, K.-H., Müller, G.: Z. Physiol. Chem. 358, 339 (1977).

37.

Bonnett, R., Gale, I. A. D., Stephenson, G. F.: 1168 (1967).

38.

Battersby, A. R., McDonald, E., Thompson, M., Bykhovsky, V. Ya.: J. Chem. Soc., Chem. Commun., 150 (1978).

39.

Murphy, M. J., Siegel, L. M., Tove, S. R., Kamin, H.: Acad. Sei. (U.S.A.) 71_, 612 (1974).

40.

For a full account of our work on sirohydrochlorin, first disclosed at the M.A.R.M. A.C.S. Meeting, Newark, Delaware, April 1977, see Scott, A. I., Irwin, A. J., Siegel, L. M., Shoolery, J. N.: J. Am. Chem. Soc. 100, 316, 7987 (1978) and references cited therein.

41.

Scott, A. I., Lee, E., Townsend, C. A.:

Hoppe-Seyler's

J. Chem. Soc. C,

Proc. Natl.

Bioorg. Chem. 3, 229 (1974).

42.

Eschenmoser, A.:

43.

Bergmann, K.-H., Deeg, R., Gneuss, K. D., Kriemler, H.-P., Müller, G.: Hoppe-Seyler's Z. Physiol. Chem. 358, 1315 (1977).

Chem. Soc. Rev. 5, 337 (1976).

44.

Battersby, A. R., McDonald, E., Morris, H. R., Thompson, M., Williams, D. C., Bykhovsky, V. Ya., Zaitseva, N. I., Bukin, V. N.: Tetrahedron Lett., 2217 (1977); Battersby, A. R., McDonald, E.: Bioorg. Chem. 7, 161 (1978).

45.

Mutant M-52. Schneider, D., Schneider, M. M., Hosozawa, S., Scott, A. I.: to be published.

46.

Scott, A. I.: Tetrahedron 31_, 2639 (1975); idem.: Soc. London, Ser. B, 273, 303 (1976).

47.

Arnold, D. P., Johnson, A. W.: (1977).

48.

Scott, A. I., Fagerness, P. E., Burton, G.: Commun. (1979) in press.

Phi los. Trans. R.

J. Chem. Soc., Chem. Commun., 787 J. Chem. Soc., Chem.

ON THE METHYLATION PROCESS IN COBYRINIC ACID BIOSYNTHESIS

G. Müller, R. Deeg, K.D. Gneuß and G. Gunzer Institut für Organische Chemie, Biochemie und Isotopenforschung der Universität Stuttgart, Pfaffenwaldring 55, D-7000 Stuttgart. H.-P. Kriemler Zentrale Funktion Forschung, Ciba-Geigy AG, CH-4-002 Basel.

After uroporphyrinogen III (uro'gen III) had been shown to be an intermediate in cobyrinic acid biosynthesis (1, 2, 3) it seemed of interest to extend the investigations beyond uro'gen III. Since cell-free extracts of Clostridium tetanomorphum and Propionibacterium shermanii can synthesize cobyrinic acid from 5-aminolevulinic acid (ALA) and S-adenosylL-methionine (SAM) (3) they were thought to be an ideal biological source for these investigations. Briefly the principles of our studies are as follows: 1. Seeking and trapping intermediates from radioactively labelled precursors with cell-free extracts and whole cell suspensions. 2. Investigations on the utilization of the trapped and radioactively labelled compounds for cobyrinic acid formation. 3. Structural elucidation of the trapped intermediates. 4-. Verification of single reactions from established steps with purified enzymes. Incubations with cell-free extracts of P^ shermanii in the presence of ALA yield the well-known porphyrins with 8-4 carboxyl substituents, but mainly coproporphyrin III. Similar incubations with cell-free extracts of C^ tetanomorphum yield uroporphyrin III (uro III) and small amounts of a hep-

© 1979 Walter de Gruyter & Co., Berlin • New York Vitamin B I 2

280 tacarboxylic porphyrin of type III (hepta). The appearance of this "hepta" was striking in particular since C ^ tetanomorphum is known to produce no haem but does make corrinoids. A heptacarboxylic uro'gen III decarboxylated in ring C (hepta 1 gen) was also considered as the next step beyond uro'gen III in cobyrinic acid biosynthesis. Several comparative incorporation experiments for cobyrinic acid formation in an usual manner (3» 4) with tetrapyrrole-free enzyme pre14 parations from tetanomorphum and the C-labelled porphyrinogens which were derived from uro III resp. the heptacarboxylic uro III by NaHg-reduction yielded insignificant incorporations for the hepta*gen (only 1#) in comparison with those from uro'gen III (which were 40$). Both porphyrins used were also synthesized with a tetrapyrrole-free en14 zyme preparation of C^ tetanomorphum from [4- C]ALA and isolated from the same incubation mixture. The tetrapyrrolefree enzyme preparations (4) used can be easily prepared by removal of tetrapyrrole carboxylic acids present in the cell-free extracts of P^ shermanii and C_j_ tetanomorphum by treatment on Sephadex DEAE A-25- This method proved to be advantageous not only for comparative incorporation experiments but also for the comparative labelling with highly specific radioactivity of derivatives from ALA. The determined incorporations of the comparative experiments made it unlikely that C-12 acetic acid decarboxylation could be the next step after uro'gen Ill-formation. This could be clearly demonstrated then by the following studies for seeking and trapping methylated intermediates of the cobyrinic acid pathway. For these investigations we have - during several series of experiments - used two incubations with cell- and Co

-free

extracts from C ^ tetanomorphum or P ^ shermanii and to both of them the cobyrinic acid precursors ALA (or uro'gen) and SAM were added. In one incubation the precursor of the corrin nucleus and in the second one the active methyl group of SAM had been radioactively labelled. After the incubation the

281

formed tetrapyrrole carboxylic acids were isolated from the enzyme preparation by an ion exchange resin (Sephadex DEAE A-25) and eluted therefrom as their methyl esters by esterification with methanol/I^SO^ (4). Thereafter the two methyl ester extracts were checked for identical compounds which had to be synthesized from both ALA and SAM. Several chromatographic purification steps led then to the detection of two partial methylated tetrapyrroles. There was a violet pigment which showed an orange fluorescence at 366 nm as well as lower amounts of a green pigment with red fluorescence. On the basis of later results concerning the number of introduced methyl groups, the green pigment was called factor I and the violet pigment factor II (3). To elucidate the question if factors I and II were of importance in cobyrinic acid biosynthesis incorporation experiments for cobyrinic acid formation with the radioactively labelled compounds (prepared by hydrolysis from their methyl esters (3)) and the tetrapyrrole-free enzyme preparations from C^ tetanomorphum were carried out. These investigations proved that factor II, and not factor I, was directly u s e d for cobyrinic acid formation (3). Later investigations however showed that a reduced form of factor I is also incorporated into cobyrinic acid (see later). The radioactively labelled factors I and II u s e d in the incorporation experiments were synthesized by the tetrapyrrole-free enzyme system, alternatively from [ 4 — ^ C ]ALA or [ M e - ^ C ] SAM resp. as can be shown in double-labelling experiments from differently labelled ALA and SAM (fundamental incorp. expts. are summarized in Table 2). Noteworthy and informative for the studies on the structural elucidation of factors I and II (see Table 1) had been two incubations for comparative double-labelling with a tetrapyrrole-free enzyme preparation from C^ tetanomorphum which gave a hint on the number of methyl groups introduced. A free incubation was used for the production of factors I and

202

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^ - d tí tí Ctí Ctí ^ aH 3 3 T Í X> - H O O O 6.5. Meanwhile, it is well established (UV/Vis, FD-mass, infrared (2, 4) and NMR (2)) that the unwanted compounds are mono- and di-^-lactones of the same kind which was already known from corrins. It had been Bykhovsky et al. (1975) who have described the first studies with these derivatives which were then designated as corriphyrins (2, 7). Most interesting in our studies with whole cells of shermanii had been the detection of an isobacteriochlorin which contains three methyl groups from L-methionine; it had been designated as factor III (4). In double-labelling experiments with different labelled ALA and L-methionine the radioactivity ratios of factor III methyl ester and sirohydrochlorin methyl ester (isolated from the same incubation) behave like 3:2 with regard to the introduced methyl groups (Table 2). Again most important informations on the structure of factor III came from PD-mass spectrometry. The molecular ion of factor III methyl ester appeared 14 mass units above that of sirohydrochlorin methyl ester at m/e 988. A mass spectrum of o factor III methyl ester derived from [methyl- H^]L-methionine proved unambiguously that the compound contains 3 methyl groups from L-methionine. It has shown the expected molecular ion at m/e 997 as well as the lower peaks at m/e 994, m/e 991 and m/e 988. The molecular ion of the Cu(II)-complex at m/e 1049 appeared 61 mass units above that of the Cu-free compound and gave evidence for two pyrrolenine nitrogen atoms

286 and two NH-groups. This fact, the characteristic isobacteriochlorin chromophore and the failure to dehydrogenate factor III methyl ester indicated that one of the three introduced methyl groups must be located at a methine bridge carbon atom. On the basis of positive incorporation experiments with factor III (derived from [ 4- 1 ^C]ALA) for cobyrinic acid formation as well as on mechanistic grounds it was assumed that the additional third methyl group of factor III should be the C-5 methyl group (4) of cobyrinic acid (see also (2, 7)). But the further and comparative incorporation experiments for cobyrinic acid formation (see Table 2, expte. 1) with double-labelled factor III and sirohydrochlorin which were synthesized from [ 2,3-^H^]ALA and [ methyl-'1 ^ClL-methionine and isolated from the same incubation had been confusing at the first sight. The determined [ ^ C / ^ H ] r a t i o s of the u s e d compounds met pretty well with 3 for factor III and 2 for sirohydrochlorin, but after their incorporation into cobyrinic acid the radioactivity ratios of the isolated cobyrinic acids had been nearly the same and behaved like 2:2. So factor III methyl ester was suspected to contain impurities of sirohydrochlorin methyl ester and the question arose if factor III actually was involved in cobyrinic acid biosynthesis. The actual utilization of factor III for cobyrinic acid formation could be rigorously proved then in the following manner. The double-labelled methyl esters of factors II and III from the experiments 2 and 3 (different radioactivity ratios of corresponding compounds!) of Table 2 were purified by usual chromatographic steps to sonstant specific radioactivity. The so purified factor III of experiment 2 was mixed with factor II from experiment 3 and factor III of experiment 3 was mixed with factor II from experiment 2. Thereafter the mixed methyl esters of factors II and III were separated by chromatography and the single compounds rechromatographed. These compounds were then (after hydrolysis) used

287

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1 o © ra •H 1 £ ft •p -d © 3 Ctí -H «H o o O O O IN O O O 00 « o ©•P h #» r> f #> © CS 5 -p > tí •H © XI •H -H S P -P (3 H "d •P ti •—* •H o R, o •d o ctí XI o o (7s OJ m © X) O O ID ID vO V ft •rl O N +> ^ ft o -d O Ctí -H Ctí U3 (X) h -H O «Hi-H •rt O o O OO o O O r ft •d P P Ctí t>5P — O M «-P >> a K « M * o •H ft ^ P. IfN CVJ tí -d > a) -P T) ITs H © -HP © •—• co VD r•d -P -P -H • »tí o ® o V PP < Ctí EH S 1 O O © ra •rt tí •H 1 S ft •rt © 3 •P «H O O o Ctí O ID T h « © ©-P p o h C^ O O #> •p « P a O ® OJ C ai M OJ o •P "ri X) OH •rl-d •P tí h > © •H ®Xl -d ® •h a S P -P ft ra-P p o® ft ® KN KN OJ (tí -p ctí -P tu o o O- IN ON h Ctí O tí fA EH -H•H H \ kD 00 C O o -d •d O vi ® ctí *> •k • • -P H XI ® ctí MC M OJ o C M OJ C •H £> -P H •H o ® >1® •P ctí ra -d -p o|H « tí ) steps in cobyrinic acid biosynthesis. A molecular weight estimation of the so far purified methylase by using calibrated Sephadex G-200 and Sepharose 6B columns gave an estimated molecular weight of about 220.000 10.000. It had an optimum pH around 7 and a Km~value of 2.17 x 10 M was obtained for SAM. Similar to most SAM dependent methyltransferases SAM:uro'gen-methyltransferase is sensitive to inhibition by S-adenosyl-L-homocysteine (SAH). Further studies with purified enzymes to realize the next step after dihydro-sirohydrochlorin formation as well as the enzymic realization of factor III formation shall answer the question if the isobacteriochlorins or their dihydro-analogues are part of the cobyrinic acid pathway.

291

References 1. Scott, A.I., Irwin, A.J., Siegel, L.M., Shoolery, J.N.: J. Am. Chem. Soc. 100, 316-318 (1978); and references therein. 2. Battersby, A.R., McDonald, E.: Bioorg. Chem. 2 , 161-173 (1978); and references therein. 3. Deeg, R., Kriemler, H.P., Bergmann, K.H., Müller, G.: Hoppe Seyler's Z. Physiol. Chem. ¿¿8, 339-352 (1977); and references therein. 4. Bergmann, K.H., Deeg, R.. Gneuß, K.D., Kriemler, H.P., Müller, G.: Hoppe Seyler's Z. Physiol. Chem. 358, 13151323 (1977); and references therein. 5. Siegel, L.M., Murphy, M.J., Kamin, H.: J. Biol. Chem. 248, 251 (1973); see also references in 1., 2. and 4.. 6. Arigoni, D., Deeg, R., Imfeld, M.: unpublished results (1978). 7. Bykhovsky, V.Ya., Zaitseva, N.I.: Prikl. Biokim. Microbiol. 6, 872-878 (1977); and references therein. 8. Weber, H.: Univ. Stuttgart (1978); unpublished results.

We thank the Deutsche Forschungsgemeinschaft for generous support of this work.

BIOGENESIS OF TETRAPYRROLE COMPOUNDS (PORPHYRINS AND CORRINOIDS), AND ITS REGULATION V. Ya. Bykhovsky Bakh I n s t i t u t e of Biochemistry, USSR Academy of Sciences, L e n i n s k i i Prospekt 33, Moscow, USSR There i s a large group of compounds in which absence the l i f e on our planet in i t s great d i v e r s i t y would not be p o s s i b l e .

This group includes

functional t e t r a p y r r o l e s - - c h l o r o p h y l l s , hemes and c o r r i n o i d s .

They are

involved in the complex process of s o l a r energy a s s i m i l a t i o n termed photosynthesis ( c h l o r o p h y l l s ) , oxido-reductive reactions and molecular oxygen t r a n s f e r (hemes) and in the c a t a l y s i s of approximately f i f t e e n biochemical reactions comprising intramolecular rearrangement of the carbon skeleton, reduction of ribonucleotides into deoxyribonucleotides, etc. ( c o r r i n o i d s )

transmethylation,

(1-15).

This s p e c i f i c role of functional t e t r a p y r r o l e s related to various aspects of the l i f e of animal, plant and bacterial organisms h i g h l i g h t s studies of t h e i r biogenesis as one of the fundamental problems of modern biochemistry. The l e a s t understood area i s biosynthesis of c o r r i n o i d s , in p a r t i c u l a r , vitamin B ^ widely used in pharmacology and elsewhere (16-19).

This com-

pound, which i s the most complicated among organic substances of nonprotein nature and has multiple f u n c t i o n s , o f f e r s a good number of quest i o n s many of which s t i l l

remain unanswered.

Even a s u p e r f i c i a l comparison of the structural formulas of c h l o r o p h y l l , heme and vitamin B ^ indicates that t h e i r central nuclei are s t r i k i n g l y similar.

I f one ignores certain d e t a i l s , i t can be stated that a l l

these

compounds are composed of four pyrrole r i n g s linked with methine bridges. However, the t e t r a p y r r o l e - 1 i k e , or as i t i s termed c o r r i n , moiety of the vitamin B , ? molecule i s reduced to a greater extent, contains seven methyl

© 1979 Walter de Gruyter & Co., Berlin • New York Vitamin BI2

291* groups o r i g i n a t i n g from methionine, and i t s r i n g s A and D are linked d i r ectly.

The basic modification of side chains - - residues of acetic and

propionic acids - - i s t h e i r amidation and not decarboxylation as in the case with chlorophyll and heme. This s i m i l a r i t y between the c o r r i n nucleus of vitamin B ^ and other t e t r a pyrroles becomes more evident i f one compares cobyrinic acid - - the simplest c o r r i n o i d so far detected - - and the f i r s t c y c l i c intermediate product - - uroporphyrinogen I I I .

tetrapyrrole

In t h i s case the basic

difference includes the absence of the methine bridge between r i n g s A and D and the presence of seven additional methyl groups in the molecule of cobyrinic acid.

The eighth methyl group i s formed as a r e s u l t of de-

carboxylation of the side chain of acetic acid at C-12 of r i n g C.

An-

other typical feature of cobyrinic acid i s occurrence of a cobalt atom in i t s molecule. A comparison of t h i s kind made i t p o s s i b l e as early as 1955 to put f o r ward a hypothesis that i n i t i a l

stages in the biogenesis of porphyrins and

c o r r i n ring of vitamin B ^ had very much in common.

Nevertheless, d i s -

s i m i l a r i t i e s were so important that even today mechanisms of the formation of the c o r r i n structure s t i l l

remain a subject of a thorough study.

Actinomycete i n v e s t i g a t i o n s c a r r i e d out in 1956-57 by Shemin et a l . (USA) demonstrated that delta-aminolevulinic acid ( • V D1—1 S 0 •ti +1 13 ïO s> rs; E

OL Ol

O (_> _J >-

DC 1— LU s: 1—1 a: h-

e 0 -W ö

CÛ z:

1—1

. S 1— (—1

>

LU Q Z UJ QLU Q U1 1 LU W SI >CQ M Z 1—1 SI •=c 1— 1—1

•z.

LU

"O e 3 O CL E O a a> 0 i. i.

>>

Q. ITS Í. +J ai

+->

>

e o

c i— o ^ O LU

e_> < o t—» z

o


c a) 01 o

u o

o > (LI a>

CL)

312

References 1. 2. 3. 4. 5.

6. 7. 8. 9. 10.

11. 12. 13. 14. 15. 16. 17. 18. 19.

20. 21. 22. 23.

Bakh A. N. Collected Works on Chemistry and Biochemistry. Moscow, USSR A c a d . S e i . P u b l . H o u s e , 1950. Kretovich W. L. Fundamentals of Plant Biochemistry. M o s c o w , Vysshaya Shkola Publ.House, 1971. Timiryazev K. A. S e l e c t e d Works on Chlorophyll and Light Assimilation by the Plant. M o s c o w , USSR Acad. Sei. Publ.House, 1948. Shlyk A. A. M e t a b o l i s m of Chlorophyll in the Green Plant. Minsk, Nauka i. Technika Publ.House, 1965. Krasnovsky A. A. T r a n s f o r m a t i o n of Light Energy During Photosynthesis. Molecular Mechanisms. 29th Bakh Lectures. M o s c o w , Nauka Publ.House, 1974. Godnev T. N. Chlorophyll: Its Structure and Synthesis in the Plant. Minsk, Byelorussian A c a d . S e i . P u b l . H o u s e , 1963. Oparin A. I. Emergence of Life on the Earth. M o s c o w , USSR Acad.Sei. Publ.House, 1957. Smith E. L. Nature, J61_, 638, 1948. Rickes E. L., Brink N. G., Koniuszy F. R., Wood T. R., Folkers K., Science, J 0 7 , 396, 1948. Hodgkin D., Pickworth C., Robertson J. H., T r u e b l o o d K. N., Prosen R. J., White J. G., Bonnet R., Cannon J. R., Johnson A. W., Sutherland L., Todd A. R., Smith E. L. Nature, " m , 325, 1955. Bukin V.N., Areshkina L.Ya. and Kutseva L.S. Uspekhi Sovremennoi Biologii, 40, 3(6), 269, 1955. Vorobeva L.I. Propionic Bacteria and Synthesis of Vitamin B,^. M o s c o w , Moscow University Publ. House, 1976. Areshkina L.Ya. Vitamin B . ? in the Animal Organism. M o s c o w , Nauka Publ. House, 1976. Smith E. Lester. Vitamin B.„. Foreign Literature Publ. House. u Moscow, 1962. Friedrich W. Vitamin B . ? und verwandte Corrinoide. Georg Thieme Verlag, Stuttgart, 19757 Waldman A.R. Vitamins in Animal Breeding. Riga, Zinatne Publ. H o u s e , 1977. Ryss S.M. Vitamins (Physiological Effect, M e t a b o l i s m , Therapy). Leningrad, Medical Literature Publ. House, 1963. Borisova T.G. and Goferman C.J. Foundations of the T e c h n o l o g y of Antibiotics and Vitamin B . ? . M o s c o w , Vysshaya Shkola Publ. H o u s e , 1967. Bukin V.N., Bykhovsky v.Ya. and Pantskhava E.S. In: Vitamin B.p and its Application in Animal Breeding. Moscow, USSR Acad. Sei. PUDI. House, 1971, p.9. Shemin D., Corcoran J . W . , Rosenblum C. and M i l l e t J.M. Science, J 2 4 , 272, 1956. Corcoran J.W. and Shemin D. Biochim. Biophys. Acta 25, 661, 1957. Schwartz S., Ikeda K., M i l l e r J.M. a n d Watson J.C. Science, 129, 40, 1959. Bykhovsky V.Ya., Zaitseva N.I. and M a n t r o v a G.V. Dokl. AN. SSSR, 157, 692, 1964.

313

24. Finogenova T.V. Biosynthesis of Vitamin B.„ and Flavins by Proactinomycetes of the Genus Nocardia. Can. Diss. Moscow State Univ., 1964. 25. Friedmann H.C. and Cagen L.M. Annual Rev. Microbiol, 24,159,1970. 26. Porra R.J. Biochim. Biophys. Acta, J07, 176, 1965. 27. Scott A.I., Townsend C.A., Okada K. and Kajiwara M. O.Am. Chem. Soc., 94, 8269, 1972. 28. Scott A.I., Georgopapadakou N., Ho K.S., Klioze S., Lee E., Lee S.L., Temme G.H., Townsend C.A. and Armitage J.M. J. Am. Chem. Soc., 97, 2548, 1975. 29. Battersby A.R., Ihara M., McDonald E., Satch F. and Williams D.C. J.Chem. Soc. Chem. Commun., 1975, 436. 30. Dauner Hans-0., Müller G. Hoppe-Seyler 1 s Z. physiol. Chem., 356, 1353, 1975. 31. Dolphin D. Bioorg. Chem., 2, 155, 1973. 32. Bray R.C. and Shemin D.J. Biol. Chem., 238, 1501, 1963. 33. Scott A.I., Townsend C.A., Okada K., Kajiwara M. and Whitman P.J. J. Am. Chem. Soc., 94, 8267, 1972. 34. Brown C.E., Shemin D. and Katz J.J., J.Biol .Chem.,248, 8015, 1973. 35. Scott A.I. Tetrahedron, 31_, 2639, 1975. 36. Friedmann H.C. Biosynthesis of corrinoids. In: "Cobalamin Biochemistry and Pathophysiology" B.M. Babior (Ed), Published by John Wiley and Sons, Inc., New York, 1975, p.77. 37. Scott A.I. , Phil. Trans. R. Soc. London, 273 B, 303, 1976. 38. Battersby A.R., McDonald E., Höllenstein R., Ihara M., Satoh F. and Williams D.C., J.Chem.Soc. Perkin I, 1977, 166. 39. Bukin V.N. and Mantrova G.V. In: Vitamin Resources and Their Use, 5, Moscow, USSR Acad.Sei.Publ. House, 1961, 32. 40. Bukin V.N. and Bykhovsky V.Ya., Mendeleev Chemistry Society Journal, ]]_, 5, 521 , 1972. 41. Bykhovsky V.Ya., Mantrova G.V., Zaitseva N.I., Prikl. Biochem. Microbiol., 5, 32, 1969. 42. Bykhovsky V.Ya., Zaitseva N.I. and Bukin V.N.,Uspekhi Biol. Chemistry Moscow, Nauka Publ. House, ^0, 199, 1969. 43. Bykhovsky V.Ya., Zaitseva N.I. and Bukin V.N., Dokl. AN SSSR, J85, 459, 1969. 44. Zaitseva N.I., Bykhovsky V.Ya. and Bukin V.N., Dokl. AN SSSR, J90,1476, 1970. 45. Bykhovsky V.Ya. and Zaitseva N.I., In: Vitamins, Naukova Dumka Publ. House, Kiev, 1970, p. 130. 46. Bykhovsky V.Ya., Zaitseva N.I. and Yavorskaya A.N., In: Vitamins, Naukova Dumka Publ. House, Kiev, 1974, p. 120. 47. Battersby A.R. and McDonald E., Biosynthesis of Porhyrins, Chlorins and Corrins. In: "Porhyrins and Metalloporphyrins". Smith K.M. (Ed.), Elsevier Scientific Publ. Company, Amsterdam, 1975, p. 61. 48. Bykhovsky V.Ya. Zaitseva N.I. and Bukin V.N. Dokl. AN SSSR, 21_1_, 970, 1973. 49. Bykhovsky V.Ya and Zaitseva N.I., Prikl. Biochem.Microbiol., ]2> 1 6 > 1977. 50. Bernhauer K., Wagner F., Michna H., Rapp P.,Vogelman H., Hoppe-Seyler's Z. Physiol. Chem. 349, 1297, 1968.

314

51. Bykhovsky V.Ya., Mantrova G.V. and Zaitseva N.I. Prikl .Biochem.Microbiol., 5, 32, 1969. 52. Müller G., Dieterle W. and Siebke G., Z. Naturforsch.,25b, 307, 1970. 53. Bykhovsky V.Ya., Zaitseva N.I. and Bukin V.N., Dokl. AN SSSR, 224, 1431, 1975. 54. Bykhovsky V.Ya., Zaitseva N.I., Umrikhina A.V. and Yavorskaya A.N., Prikl. Biochem. Microbiol. J_2 825, 1976. 55. Treibs A., Ann. N.Y. Acad. Sei., 206, 97, 1973. 56. Gurinovich G.P., Sevchenko A.N. and Solovyov K.N., Spectroscopy of Chlorophyll and Related Compounds. Minsk. Nauka i Technika Publ. House, 1968. 57. Bykhovsky V.Ya. and Zaitseva N.I., Prikl. Biochem. Microbiol. YZ, 365, 1976. 58. Battersby A.R., McDonald E., Morris H.R. Thompson M., Williams D.C., Bykhovsky V.Ya., Zaitseva N.I. and Bukin V.N., Tetrahedron Letters, 1977, 2217. 59. Deeg R., Kriemler H.-P., Bergmann K.-H. and Müller G., Hoppe-Seyler's Z. Physiol. Chem., 358, 339, 1977. 60. Bergmann K.-H., Deeg R., Kriemler H.-P. and Müller G., Hoppe-Seyler's Z. Physiol Chem., 358,1315, 1977. 61. Battersby A.R., McDonald E., Thopson M. and Bykhovsky V.Ya., J.Chem.Soc. Chem. Commun., 1978, 150. 62. Bykhovsky V.Ya. and Zaitseva N.I., Prikl. Biochem. Microbiol., J 3 , 872, 1977. 63. Bykhovsky V.Ya., Zaitseva N.I., Gruzina V.D., Malyarova Z.A., Ponomareva G.M. and Yavorskaya A.N., Prikl. Biochem.Microbiol.11,179,1975. 64. Scott A.I., Irwin A.J., Siegel L.M. and Shoolery J.N., J.Am.Chem.Soc. ^00, 316, 1978. 65. Battersby A.R., Jones K., McDonald E., Robinson J.A. and Morris H.R., Tetrahedron Letters, 1977, 2213. 66. Siegel L.M., Murphy M.J. and Kamin H., J.Biol.Chem.,248, 251, 1973. 67. Murphy M.J., Siegel L.M., Kamin H. and Rosental D., J.Biol.Chem., 248, 2801 , 1973. 68. Murphy M.J. and Siegel L.M., J.Biol.Chem., 248, 6911, 1973. 69. Porra R.J. and Skyring G.W., In: "Porphyrins in Human Diseases". Doss M (Ed.), Publ. S.Karger, Basle, 1976, p. 459. 70. Postgate J.R., J.Gen. Microbiol., U , 545, 1956. 71. Myasishcheva N.V., Vitamin B ^ Content in Blood of Healthy Men and Patients with Certain Hematological and Surgical Diseases. Cand. Diss., Leningrad, 1960. 72. Kubatiev A.A., Porphyrins, Vitamin B.„ and Cancer. Priokskoye Publ. House, Tula, 1973. 73. Poznanskaya A.A., Vitamin B.^» In: Vitamins, Medizina.Publ.House, Moscow, 1974. 74. Andreeva N.A., Enzymes in Metabolism of Folic Acid. Nauka Publ. House, Moscow, 1974.

F a c t o r I ex C l o s t r i d i u m t e t a n o m o r p h u m : ture a n d R e l a t i o n s h i p by M. I m f e l d a n d D.

to V i t a m i n B ^

P r o o f of S t r u c -

Biosynthesis

Arigoni,

L a b o r für o r g a n i s c h e C h e m i e , E T H Z ü r i c h , R. D e e g a n d G. Institut

and

Müller,

für o r g a n i s c h e

Chemie, Biochemie

Isotopenforschung, Universität

und

Stuttgart.

F a c t o r I was first o b t a i n e d as o c t a m e t h y l e s t e r t o g e t h e r

with

the e s t e r of f a c t o r 11"^ ( i d e n t i c a l w i t h s i r o h y d r o c h l o r i n u p o n i n c u b a t i o n of u r o ' g e n I I I , 1, w i t h a cell free of the title o r g a n i s m .

preparation

Its s t r u c t u r e w a s t e n t a t i v e l y

formulated

as in 2 on the b a s i s of the a v a i l a b l e a n a l y t i c a l data. In rast to

)

cont-

f a c t o r I is not c o n v e r t e d to c o b y r i n i c a c i d , 5_. It

has now b e e n f o u n d w i t h d i f f e r e n t l y

radiolabelled

substrates

that a r e d u c e d f o r m of f a c t o r I, a v a i l a b l e t h r o u g h p

P

1

A

P

P

P

A

2

P

5

4

3

A: CHjCOJH

© 1979 Walter de Gruyter & Co., Berlin • New York Vitamin Bis

P : CH2CHJC02H

reduction

316

with Na/Hg, and probably identical with the tetrahydroderivative _3, is efficiently incorporated by cell preparations of CI. tetanomorphum both into sirohydrochlorin,

and cobyrinic

acid, 5. The position of the label in the cobester specimen, 6, obtained from

^nethyl-^c]-factor I, was established through

the following degradation:

*Me: 14CH3 These results indicate that (i) the structure of factor I is correctly formulated as in 2_, (ii) a reduced form of 2_, probably the tetrahydroderivative _3, is a precursor of both sirohydrochlorin,

and cobyrinic acid, 5, on the way to vitamin

B^^, (iii) the methyl group of 3 retains its position at C-2 during the formation of

The same must be true for the

corresponding methyl group of sirohydrochlorin. (1)

R. Deeg, H.-P. Kriemler, K.-H. Bergmann, and G. Muller, Z. physiol. Chem., 1977, 358, 339-

(2)

A.R. Battersby, E. McDonald, M. Thompson, and V. Ya. Bykhovsky, J.C.S. Chem. Comm., 1978, 150.

(3)

A.I. Scott, A.J. Irwin, L.M. Siegel, and J.N. Shoolery, J. Amer. Chem. Soc., 1978, 100, 316.

ON THE BIOSYNTHESIS OF THE 5 ,6-DIMETHYLBENZIMIDAZOLE OF VITAMIN

MOIETY

B12

P. Renz, J. Hörig and R. Wurm Institut f ü r Biologische Chemie, Universität D - 7 0 0 0 Stuttgart 70, West Germany

Hohenheim

The n a t u r a l l y occurring base-containing corrinoids can be d i v i d e d into three groups: those containing a benzimidazole base, those w i t h a purine base, a n d those w h i c h contain a nitrogen-free compound like phenol or p-cresol b o u n d O - g l y c o sidically to ribose (1). In the g r o u p of corrinoids

contai-

ning a benzimidazole base the following bases are found: benzimidazole

(I), 5 - m e t h o x y b e n z i m i d a z o l e

benzimidazole

(III), 5-methoxybenzimidazole

methylbenzimidazole

(II), 5 - h y d r o x y -

(V) and naphthimidazole

(IV), 5 , 6 - d i (VI). I, II and

V I were isolated from activated sludge. They are f o r m e d u n d e r aerobic conditions. Ill is f o r m e d by methane

bacteria

a n d mainly found in the anaerobic sewage sludge (1). IV is f o r m e d by Clostridium thermoaceticum (2), an obligate

an-

aerobe. Preliminary experiments on the biosynthesis of IV s h o w e d that III and 5 - h y d r o x y b e n z i m i d a z o l y l c o b a m i d e

are

readily m e t h y l a t e d by Clostridium thermoaceticum (5) w i t h methionine as methyl g r o u p donor. Methionine is also the methyl g r o u p donor of the methoxy methyl group of IV in the de novo biosynthesis of 5 - m e t h o x y b e n z i m i d a z o l y l c o b a m i d e

(4).

We do not yet know how the 5-hydroxybenzimidazole part is formed, but we found that shikirnic acid is not a precursor of III. Also the b i o s y n t h e s i s of I, II and V I is not known. 5,6-dimethylbenzimidazole, the base of vitamin B,^» f o r m e d by aerobic, aerotolerant and anaerobic

microorgan-

isms (5,6). Some of them, w h i c h were u s e d in this

© 1979 Walter de Gruyter & Co., Berlin • New York Vitamin Bis

is

laboratory

31B f o r the investigation of the b i o s y n t h e s i s of 5>&-diniethylbenzimidazole, are l i s t e d in Table 1. Table 1

Some Microorganisms Forming V i t a m i n

B ^

(5,6-dimethylbenzimidazolylcobamide) Aerotolerant A e r o b e s

A n a e r o b e s

A n a e r o b e s

Bacillus

Propionibacterium shermanii

Butyribacterium rettgeri

Propionibacterium freudenreichii

Clostridium

megaterium

Streptomyces

sp.

barkeri

Nocardia r u g o s a

P. shermanii and P. f r e u d e n r e i c h i i form

5>6-dimethylbenzimi-

dazole only under aerobic conditions. This shows that

oxygen

is r e q u i r e d for the b i o s y n t h e s i s of this base. We f o u n d that 5,6-dimethylbenzimidazole

is f o r m e d in P. shermanii from

r i b o f l a v i n (7) and that C - 1 ' of the ribityl side chain is t r a n s f o r m e d into C - 2 of 5,6-dimethylbenzimidazole recently we could demonstrate that

(8). More

5,6-dimethylbenzimidazole

is formed in the same way in P. f r e u d e n r e i c h i i and in the aerobic microorganisms m e n t i o n e d in Table 1. The s

anaerobic

n

v i t a m i n B^2~ y ~khesizing microorganisms m e n t i o n e d in Table 1 do not use riboflavin as p r e c u r s o r of the

5>6-dimethylbenz-

imidazole moiety of v i t a m i n B,^. The experiments w i t h P. shermanii and P. f r e u d e n r e i c h i i were carried out in "che following way: the bacteria were grown anaerobically in the presence of cobalt. Thus mainly amide is formed. The cells were then h a r v e s t e d ,

cobin-

suspended

in 0.06 M phosphate b u f f e r (pH 7.0) a n d incubated a e r o b i cally in the presence of riboflavin. By this aerobic bation the cobinamide is transformed into v i t a m i n

incu-

B„T

319

A l t h o u g h it was clearly shown w i t h radioactively

labeled

r i b o f l a v i n that this compound is the precursor of 5 > 6 - d i m e t h y l b e n z i m i d a z o l e , we could n e v e r detect free benzimidazole

^jfe-dimethyl-

in bacteria grown in the presence of cobalt,

but only as a component of the v i t a m i n Therefore we w a n t e d to know if free

B^2_m°lecule•

5,6-dirnethylbenzimid-

azole is a c t u a l l y occurring during the b i o s y n t h e s i s of 15 v i t a m i n B ^ * I11 order to answer this question 5 - N - r i b o f l a v i n was chemically synthesized and added to our v i t a min B ^ - s y n t h e s i z i n g P. shermanii-system. If free methylbenzimidazole of v i t a m i n

5,6-di-

is an intermediate in the b i o s y15 nthesis

the two nitrogens s h o u l d be equally

beled. If 5,6-dimethylbenzimidazole

^N-la-

does not occur freely

e i t h e r N-1 or N - 3 of the 15 5>6-dimethylbenzimidazole moiety of v i t a m i n B ^ 0 should be ^N-labeled. In order to determine 15 the distribution of the ^N-label the v i t a m i n B,^ w a s d e g r a d e d to 0(-ribazole

(5,6-dimethylbenzimidazole-Qi-D-ribo-

furanoside) a n d a c e t y l a t e d to its triacetyl derivative. The tri-0-acetyl-06-ribazole was e x a m i n e d for the d i s t r i b u 15 t i o n of the ^N-label by proton magnetic resonance s p e c t r o scopy (9). The result is shown in Figure 1. It represents that part of the 90 MHz

H M R - s p e c t r u m which is caused by

the C - 2 - p r o t o n of 5 > 6 - d i m e t h y l b e n z i m i d a z o l e . The peak a s s i g n e d H - 2 is the singlet of the C - 2 - p r o t o n of u n l a b e l e d molecules r m ethe d from endogenous 15 N-1 is due f oto coupling of the riboflavin. C - 2 - p r o t o n The w i t h doublet -^N located at N - 1 , and the doublet N - 3 is caused by the c o u p 15 ling of the C - 2 - p r o t o n w i t h ^N l o c a t e d at N - 3 . P r o m the different peak heights resp. integrals it can be

calculated

that 27 per cent of 15 the molecules are u n l a b e l e d , and that 6 0 per cent of the ^N-label is l o c a t e d at N-1 a n d 40 per cent at N - 3 . This asymmetric distribution of

15 ^N-label b e t w e e n N-1 and

N - 3 shows that 80 per cent of the

5>6- 6 - d i m e t h y l b e n z i m i d a z o l e unit of tri-0-acetyl-o(-ribazole d e r i v e d from v i t a m i n B ^ F o r f u r t h e r d e t a i l s see t e x t a n d r e f e r e n c e (9)

m o l e c u l e s f o r m e d d u r i n g the b i o s y n t h e s i s of v i t a m i n B,^ g e t into a free state. B u t 2 0 p e r cent of the

5»6-dimethylbenz-

imidazole m o l e c u l e s m i g r a t e to the n e x t enzyme in the

bio-

s y n t h e t i c p a t h w a y , the t r a n s - N - g l y c o s i d a s e f o u n d by F r i e d m a n n (10), w i t h o u t l o s i n g t h e i r a s y m m e t r y . T h i s is t e d in F i g u r e

illustra-

2.

The fact that 20 p e r c e n t of the

5,6-dimethylbenzimidazole

m o l e c u l e s do not lose t h e i r a s y m m e t r y i m p o s e d by the 15 cursor 5 -

pre-

N - r i b o f l a v i n g i v e s indirect proof t h a t the

enzyme s y s t e m t r a n s f o r m i n g r i b o f l a v i n into

5,6-dimethylbenz-

321

(Trans-N-glycosidase) Figure 2

A s s o c i a t i o n of the enzyme system of P. shermanii transforming riboflavin into 5 , 6 - d i m e t h y l b e n z imidazole, a n d the trans-N-glycosidase as r e v e a l e d 15 by the experiments w i t h 5 - N - r i b o f l a v i n a n d the N M R - m e a s u r e m e n t s d e s c r i b e d in the text.

imidazole a n d the trans-N-glycosidase must be very closely a s s o c i a t e d in the intact bacterial cell. A l t h o u g h we were not able to decide w i t h normal methods of analytical b i o c h e m i s t r y , if 5>&-dimethylbenzimidazole

occurs

in free form during the v i t a m i n B^, 0 - b i o s y n t h e s i s , the prob15 lem was solved by the use of ^N-label and the a p p l i c a t i o n 1 of HMR-spectroscopy. It is tempting to speculate that not only the two enzyme systems m e n t i o n e d in Figure 2 are in close v i c i n i t y to each other as f o u n d here, but that the whole enzyme system of the v i t a m i n

biosynthesis

is

a r r a n g e d as a multienzyme complex in the intact bacterial cell. Acknowledgement: The financial support of this work by the Deutsche F o r s c h u n g s g e m e i n s c h a f t

is gratefully

acknowledged.

322 References u n d

(1) F r i e d r i c h , W.: V i t a m i n B ^

verwandte

Corrinoide,

in Ammon, R. u. D i r s c h e r l , W. ¡Fermente, Hormone, V i t a mine, B a n d III/2, S. 29 - 4 - 1 , G. T h i e m e , S t u t t g a r t , 1975(2) Irion, E . , and Ljungdahl, L.G.: Biochemistry

2780-2790

(1965).

(3) W u r m , R., W e y h e n m e y e r , R., a n d Renz, P.: Eur. J. Biochern. ¿6, 4 2 7 - 4 3 2

(1975).

(4-) Wurm, R., and Renz, P.: unpublished. (5) see ref. (1) p. 1 7 8 - 1 8 0 and 2 0 5 - 2 0 6 . (6) Perlman, D., and Semar, J.B.: Biotechnol. Bioeng. 5, 2 1 - 2 5 (1963) (7) Renz, F.: F E B S - L e t t e r s 6, 187-189 (8) Renz, P., and W e y h e n m e y e r , F E B S - L e t t e r s 22, 124-126

(1970).

R.:

(1972).

(9) Hörig, J., Renz, P., and Heckmann, G.: J. B i o l . Chem. 2£3, 74-10-7414- (1978). (10)Friedmann, H.C.: J. Biol. Chem. 240, 413-4-18 (1965).

THE ENZYME SYSTEM OF PROPIONIC ACID BACTERIA TRANSFORMING RIBOFLAVIN INTO 5,6-DIMETHYLBENZIMIDAZOLE

J. Hörig & P. Renz Institut für Biol. Chemie der Universität Hohenheim, 7-Stuttgart-?0, Garbenstr. 30, FRG

Introduction In experiments with Propionibacterium shermanii it was shown that the 5,6-dimethylbenzimidazole moiety of vitamin B i s formed from riboflavin. Thereby the C-1' of the ribityl side chain of riboflavin is transformed into the C-2 of DBI as demonstrated with 1'-1^C-riboflavin (1). Recently we found that cells of Propionibacterium freudenreichii, grown without a cobalt salt in the growth medium and thus containing only trace amounts of corrinoids, form free DBI and «k-ribazole from riboflavin (2). Subsequently we studied the formation of DBI from riboflavin in greater detail using broken cell preparations of P. freudenreichii and P. shermanii. These experiments are described in this paper.

Materials and Methods 1 ' ^ - R i b o f l a v i n (289 000 dprn/ztmol) was synthesized according to Kuhn et al. (3) starting from 1 mmol of 1-^C-ribose. Propionibacterium shermanii St 33 and Propionibacterium freudenAbbreviations: DBI, 5,6-dimethylbenzimidazole; oC-ribazole, 5,6-dimethylbenzimidazole-oi.-D-ribofuranoside; o(-ribazole 5'phosphate, 5,6-dimethylbenzimidazole-a(.-D-ribofuranoside 5'phosphate; FMN, flavin mononucleotide.

© 1979 Walter de Gruyter & Co., Berlin • New York Vitamin B l t

324 reichii ATCC 6207 were grown anaerobically as described earlier (1+), but cobaltous sulfate was omitted from the growth medium. The P. shermanii-cells were centrifuged for 15 min. at 7 000 x g and directly used for the experiments described below. The P. freudenreichii-cells were centrifuged for 60 min at 7 000 x g. The slimy sediment was.resuspended in an equal volume of 0.9% NaCl and centrifuged for 60 min at if8 000 x g. The cell paste of both organisms was stored at -20°. 20 - 40 g of frozen cells were pressed at -20° three times through a X-press (AB Biox, Nacka, Sweden). One part of the broken cell mass was mixed with two volumes of 0.3 M phosphate buffer pH 6.5 (390 mM Na + , 16,5 mM K + , 1 mM MgCl2). A small amount (20 ^g/ml) of DNAse I (Boehringer, Mannheim) was added. 9.5 nil of this homogenate was pipetted into 25 ml-Erlenmeyer flasks and 0.5 ml (400 nmol) of an aqueous solution of riboflavin added. The mixture was incubated with shaking for 16 hrs at 30°. Then 0.5 ml of glacial acetic acid and 1 ml of a solution of 2-^C-DBI (0.15>imol, 39 600 dpm/)imol) were added. The mixture was heated (120°, 10 min), cooled and centrifuged. The supernatant was brought to pH 10 - 12 and extracted with 15 ml of chloroform. The chloroform was evaporated. The residue was redissolved in a small volume of chloroform and subjected to thin layer chromatography on silica gel with CHCiyEtOH/AcOH = 85/15/1. The DBI-band was scraped off and the DBI eluted with 2 ml of EtOH/ conc. NH^ = 10/0.5. From the specific radioactivity of the DBI thus obtained the absolute amount of DBI formed could be calculated.

Results and Discussion P. freudenreichii cells were grown without a cobalt salt in the medium in order to minimize corrinoid formation. A homogenate of such cells transforms 1'-1^C-riboflavin into free radioactive DBI as shown in Figure 1. This experiment demonstrates that

325

œ a

Figure 1 : Evidence for the transformation of 1'- ^C-riboflavin into DBI in a cell-free preparation of Propionibacterium freudenreichii. Radioactivity scanner trace of a thin layer chromatogram of the chloroform extract of the incubation mixture. 60 ml of homogenate was incubated with 0.9 /imol 1 11 ^-riboflavin for 16 hrs. The two minor peaks are heat destruction products of radioactive riboflavin formed during the isolation procedure. cell homogenates of P. freudenreichii are suitable for the investigation of some properties of the DBI-forming enzyme system. Thus we could show that the pH-optimum of the DBI-formation is pH 6.5. At this pH the DBI formed is not further metabolized. This was demonstrated with exogenously added DBI. The reason for this effect is that the next enzyme in the biosynthetic pathway, the trans-N-glycosidase is almost inactive at pH 6.5. The pH-optimum for the latter enzyme, transforming DBI into «t-ribazole 5'-phosphate, lies near pH 9 (5). In the course of our experiments we were able to demonstrate that the yield of DBI is very sensitive to variations in oxygen concentration in our homogenate system. From a homogenate pool containing all ingredients inclusively riboflavin samples from

326

5

10

15

[ml] Volume of sample

Figure 2: Oxygen dependence of the DBI-formation from riboflavin in the cell-free preparation of Propionibacterium freudenreichii. Different volumes of homogenate containing kO nmol/ml riboflavin were pipetted into 25 ml Erlenmeyer flasks and incubated for 16 hrs. 3 to 15 ml were pipetted into 25 ml Erlenmeyer flasks and incubated for 16 hrs.

As shown in Figure 2 the yield of DBI has

a sharp maximum in the Erlenmeyer flask containing the 10 ml sample. The existence of such an "optimal volume" may be explained in the following way: the first step in the transformation of riboflavin into DBI is the reduction of riboflavin or a phosphorylated derivative of it. Molecular oxygen is then covalently attached to the reduced flavin. At the higher oxygen concen-

327

P. freudenreichii P. shermanii

200

100'

10

20

30 ImM] Nicotinamide

Figure 3: Stimulation of the DBI-fomation in cell-free preparations of Propionibacterium freudenreichii and P. shermanii by nicotinamide. 9.5 ml of homogenate was incubated~for lb firs with ¿+00 nmol riboflavin in the presence of the given concentrations of nicotinamide. tration present when small volumes are incubated, the reduced flavin-oxygen complex readily decomposes nonenzymatically into hydrogen peroxide and the oxidized form of

the flavin. Thus only

a small amount of reduced flavin is available to the DBI-synthase and the reducing power of the sample necessary for the DBIformation is quickly exhausted. At the lower oxygen concentrations present when higher volumes are incubated, the DBI-synthase may not be maximally active because of the lack of oxygen. Another property of the P. freudenreichii-homogenate is shown in Figure 3. We found that the DBI-formation in homogenates of cobalt-free grown P. freudenreichii as well as in homogenates of P. shermanii is stimulated by nicotinamide. Nicotinamide can be substituted by nicotinic acid, which produces the same effect. This is most probably due to the rapid conversion of nicotinamide to nicotinic acid in homogenates of P. freudenreichii. The results may be explained by an allosteric activation

328

CH20-P03' (HOCH)2 HOCH

CH2O-PO3" (HOCH)2

,0

C C

©

Erythrose '»-phosphate

© •ft

• m 5,6-OBI

Figure Hypothetical scheme for the transformation of FMN into DBI by propionic acid bacteria. of the DBI-synthase by nicotinic acid or more likely by a derivative of it. This derivative may be nicotinic acid mononucleotide being the cosubstrate of the trans-N-glycosidase forming et-ribazole 51-phosphate. Thus in the intact cell DBI would be only formed in the presence of nicotinic acid mononucleotide, its reaction partner for the next biosynthetic step. The activating effect of nicotinic acid mononucleotide may be mediated by the trans-N-glycosidase, because the DBI-synthase and the trans-N-glycosidase are located in close vicinity to each other in the bacterial cell (6). Furthermore, in intact cells of P. freudenreichii and P. shermanii the biosynthesis of DBI must be regulated by corrinoids. This is shown by the fact that intact cobalt-free grown cells of P. shermanii produce only trace amounts of DBI when incubated with riboflavin (2). But the same organism produces large amounts of DBI (as vitamin B 2 (6)), when it is grown in the presence of a cobaltous salt thus producing corrinoids.

329

In Figure k a hypothetical scheme for the transformation of riboflavin into DBI is shown. We have preliminary data supporting some of the reactions mentioned in this scheme. In some of our experiments we observed a more rapid formation of DBI using FMN instead of riboflavin as substrates. Therefore we assume that FMN is the real substrate. The first reaction, the reduction of flavins in an extract of P. freudenreichii, can be readily observed spectrophotometrically at 4 50 nm. Although we could not yet identify erythrose ¿^-phosphate as a reaction product of the C-2' to C-51 of the ribityl side chain of riboflavin (reaction 3), we were able to isolate a mixture of radioactively labeled sugar phosphates. DBI is readily formed nonenzymatically from 1,2-diamino-/f,5dimethylbenzene and formaldehyde (7). In this reaction the N-methylenephenylenediamine mentioned in this scheme (reaction 5) may be an intermediate. Acknowledgement: The financial support of this work by the Deutsche Forschungsgemeinschaft is gratefully acknowledged.

References 1. Renz, P., Weyhenmeyer, R.: FEBS-Letters 22, 1-2/+ - 126 (1972) 2. Hörig, J., Renz, P.: FEBS-Letters 80, 337 - 339 (1977) 3. Kuhn, R., Ströbele, R.: Ber. dtsch. ehem. Ges. 70, 773 778 (1937) k. Renz, P.: Meth. in Enzymol. 18c, 82 - 92 (1971) 5. Friedmann, H.C., Harris, D.L.: J. Biol. Chem. 2^0, i+13 ¿fl8 (1965) 6. Hörig, J.A., Renz, P., Heckmann, G.: J. Biol. Chem. 253, 7A-10 - 7¿fH (1978) 7. Renz, P., Wurm, R., Hörig,J.: Z. Naturforsch. 32c, 523 527 (1977)

STRAIGHT

Herbert

APPROACHES

C.

TO THE N U C L E O T I D E

LOOP

Friedmann

Department of B i o c h e m i s t r y , The U n i v e r s i t y of Chicago 920 East 58th S t r e e t , Chicago, I l l i n o i s 60637, U.S.A.

Introduction Most of the b i o s y n t h e t i c p r e s e n t a t i o n s at t h i s meeting deal with the beg i n n i n g s of an i n s i g h t i n t o the formation of the complex c o r r i n r i n g tem.

sys-

Much of t h i s d i f f i c u l t work has been preceded by i n v e s t i g a t i o n s on

the formation o f the other and simpler r i n g system of cobalamin, namely of the s o - c a l l e d nucleotide l o o p , a s t r u c t u r e which i s a l s o a c o l l e c t i o n of chemical o d d i t i e s . nature to B ^ .

Thus the base, 5,6-dimethylbenzimidazole, unique in

i s the only p h y s i o l o g i c a l

o-xylene d e r i v a t i v e besides

ribo-

f l a v i n and i t i s , i n f a c t , as we have seen e a r l i e r , b i o s y n t h e t i c a l l y

rela-

ted to t h i s vitamin.

Long before the base-on and b a s e - o f f

relationships

between base and cobalt were recognized, two other s t r u c t u r a l

features

connected with the base were known, namely the prevalence of a v a r i e t y of bases other than 5,6-dimethylbenzimidazole in many analogs of the v i t a m i n , and the l i n k a g e of these bases to nucleotide r i b o s e by an N - a - g l y c o s i d i c bond, rather than by the N - 6 - g l y c o s i d i c bond found in n u c l e i c a c i d s and i n the common n u c l e o t i d e s .

We s h a l l see s h o r t l y that the v a r i a b i l i t y in the

bases and the constancy o f the N - a - g l y c o s i d i c bond have a common enzymatic, biosynthetic origin.

As one proceeds f u r t h e r along the n u c l e o t i d e l o o p ,

one d i s c o v e r s a phosphodiester with two somewhat unusual f e a t u r e s : i t comp l e t e s a 3' rather than a 5 ' - n u c l e o t i d e , and i t s other attachment i s to the rare ( R ) - l - a m i n o - 2 - p r o p a n o l

moiety.

I s h a l l here d i s c u s s the f o l l o w i n g

three t o p i c s : (1) the enzymatic b a s i s f o r the formation of the N - a - g l y c o s i d i c bond and f o r the i n c o r p o r a t i o n of v a r i o u s bases i n N - a - g l y c o s i d i c

link-

age; (2) a b i o s y n t h e t i c consequence o f the attachment of phosphate to the 3' carbon of the r i b o s e moiety; and (3) the formation of

(R)-l-amino-2-pro-

panol from L - t h r e o n i n e , and the attachment of t h i s base to the p o s i t i o n £

© 1979 Walter de Gruyter & Co., Berlin • New York Vitamin BI8

332 propionic acid side chain in ring D of the corrin system.

Formation

of the

N-a-Glycosidic

Bond

This work was done some time ago (see 1, 2) but it will be presented here briefly since it initiated my interest in the B ^ the time of the last B ^

Symposium in 1961.

field and was unknown at

When I became interested in

this topic in the early sixties three important facts were known: first, as already mentioned, a number of naturally occurring corrinoids contained bases other than 5,6-dimethylbenzimidazole, and, when the microbiologist rather than the microorganism added the base, bacterial cultures could incorporate a large variety of bases into the nucleotide to form numerous B-J2 analogues (see for example 3, and references quoted in 1 and 2).

The

latter phenomenon, known as guided biosynthesis, had been extensively studied in the hope of discovering a B-^ analogue that might have a n t i - B ^ and hence chemotherapeutic activity, a hope that was never realized. Second, a number of so-called incomplete corrinoids, i.e. molecules lacking the base and other parts of the nucleotide loop were known to occur in bacterial cultures, namely cobyric acid and cobinamide, i.e. the fully amidated corrinoids respectively lacking and containing the ((R)-l-amino2-propanol moiety, as well as cobinamide phosphate (4-9) and, of most importance for the present studies, yet another corrinoid (7-9), the activated form of cobinamide phosphate, namely guanosine diphosphate cobinamide (10-13).

Third, it had been shown by means of labeling studies that,

contrary to the conclusion (14, 15) that the free base was directly incorporated into a cobamide, the base had to be attached to ribose or to a ribose-containing compound before its incorporation into corrinoid (16). The function of guanosine diphosphate cobinamide as a biosynthetic precursor (12, 17, 18, see also 19 and 20) would of course have made very little sense if base was to be incorporated into corrinoid in one step.

It is of

interest that one laboratory, that of DiMarco in the firm of Farmitalia in Milan is responsible for the identification of guanosine diphosphate cobinamide and for the labeling experiments. In initial experiments I was able to show that when benzimidazole is given

333 to a suspension of Propionibacterium shermanii the corresponding nucleoside appears in the medium.

This could be isolated by chromatography on a DEAE-

cellulose column after extraction into isobutanol and removal of free benzimidazole as silver salt (21).

It was soon found that biosynthetically the

5'-nucleotide rather than the nucleoside is first formed by a novel transN-glycosidase or phosphoribosyltransferase which catalyzes the single-displacement (or what the organic chemist would call an S^2) reaction between free base and the ribose phosphate moiety of nicotinic acid 5'-mononucleotide (22-24): Nicotinate-g-R-5 1 -P +

+

Base

>

Base-a-R-5'-P

+

Nicotinate

+

H+

This reaction is driven by the conversion of the quaternary bond between nicotinate and ribose to the uncharged new base-ribose bond, with the associated liberation of a proton.

It was easy to show that the compound for-

med was N-a-glycosidic, since an extract of Lactobacillus delbrueckii, known to contain a hydrolase specific for N-B-glycosidic nucleosides (25, 26) did not attack the nucleoside formed from the nucleotide by dephosphorylation while it did cleave the synthetic N-6-glycosidic benzimidazole nucleoside (22, 24).

The discovery of this trans-N-glycosidase explained

not only the formation of the N-a-glycosidic bond but also the existence of guided biosynthesis, since the enzyme was rather unspecific for the type of base utilized.

Moreover, this specificity appeared to parallel

the specificity for incorporation of base observed in vivo with different microorganisms.

Thus, 2-methylbenzimidazole, which is not incorporated

into corrinoids (14), was not a substrate (23), and while the enzyme from shermanii appeared to act weakly if at all with adenine (23), the enzyme from Clostridium sticklandii catalyzed a trans-N-glycosidation both with benzimidazoles and with adenine (24).

The reaction of the enzyme from the

latter organism with adenine was most interesting: adenine, in contrast to 5,6-dimethylbenzimidazole, is an asymmetric compound in the sense that the two nitrogens of its imidazole moiety are not equivalent to each other, and in fact a novel analogue of the common 5'-adenylic acid was formed by this enzymatic reaction: not only was this enzymatically prepared new adenine 5'-nucleotide a-glycosidic, but, as shown by absorption spectrum, by instability and by chromatographic behavior, it was substituted at N^ rather than at N Q (27).

This substitution was exactly the one that had been de-

331*

monstrated by X-ray crystallography (28) and by chemical isolation (29) to be the one that obtains in ip-vitamin B ^ .

The formation of this nucleotide

hence confirmed the role of the trans-N-glycosidase in corrinoid biosynthesis.

This i s the only enzyme known up to now to produce a compound with an

N-a-glycosidic bond.

In the course of these studies a novel deamidase was

discovered which converts nicotinamide mononucleotide to nicotinate mononucleotide and which does not attack the nucleoside or free nicotinamide (24, 30, 31).

This enzyme has been purified to homogeneity.

F o r m a t i o n of V i t a m i n B ^ of the

5 1 - P h o s p h a t e , the Immediate

Precursor

Vitamin

Since the phosphate in the nucleotide loop of corrinoids i s attached to the 3' rather than to the 5' carbon of the ribose, and since i t had already been shown by the group in Milan that i t is the phosphate of cobinamide phosphate and the corresponding inner phosphate of guanosine diphosphate cobinamide that occurs in the vitamin (32), an immediate question was posed by the enzymatic formation of the 5'-nucleotide of various bases: i s i t the 5'-nucleotide that i s incorporated, or is this nucleotide dephosphorylated to the nucleoside before incorporation?

In the f i r s t but not

in the second case one would expect to find a phosphorylated corrinoid. Such a substance would be more negatively charged than the corresponding form of the vitamin.

When a suspension of V_. shermanii was incubated with

-dimethylbenzimidazole, an extract of the bacteria, analyzed by ionophoresis after photolysis and after cyanide addition, did indeed show two and only two labeled corrinoid regions.

One of these manifested

the behavior of aquo- or of cyanocobalamin, while the mobility and chromatographic behavior of the other in the presence of cyanide was identical to that of cyanocobalamin 5'-phosphate (33, 34).

This substance had been

synthesized (35), following a published outline (36) by reaction under anhydrous conditions between cyanocobalamin and e-cyanoethyl phosphate in the presence of dicyclohexylcarbodiimide, followed by slow ammoniacal hydrolysis of the product at a low temperature.

The amount of the corres-

ponding bacterial substance was small, of the order of half a mg per kg wet weight bacteria.

However, an

increase in t h i s compound (33, 34), up

335 to 40-fold (37, 38), could be obtained by the simple expedient of a slight heat-treatment of the bacteria under narrowly defined conditions.

It ap-

peared that this fortuitous effect was due to a greater heat-sensitivity in the further metabolism than in the formation of this compound.

With this

expedient it became possible, using Dowex 50 followed by DEAE-cellulose chromatography, to isolate this substance in pure form for crystallization and X-ray crystallographic investigation (37, 39).

Kinetic experiments

demonstrated this substance to be an obligatory intermediate in cobalamin biosynthesis (34).

Chemically a novel nucleotide was prepared from it

which contained one more phosphate than the nucleotide (a-ribazole 3'-phosphate) obtainable by acid hydrolysis of the vitamin.

The extra phosphate

was shown to be located at the 5' position of the ribose. mediate was concluded to be cobalamin 5'-phosphate.

The new inter-

One can say, strictly

in terms of chemistry, and ignoring enzyme preferences and structural constraints, that the function of the phosphate group in a-ribazole 5'-phosphate is to ensure the corrinoid nucleotide loop not to contain a 5'-phosphate: the a-ribazole 5'-phosphate may be regarded as containing a blocked primary alcohol group which is hence protected from attack by the inner phosphate of guanosine diphosphate cobinamide. The biochemical behavior of cobalamin 5'-phosphate caused some difficulties: it had been found that the 5' phosphate group of the new corrinoid is not hydrolyzed by alkaline phosphatase. this phosphate group was well exposed.

The X-ray data indicated that

In work carried out with Zenon

Schneider in my laboratory (40) it was found that Escherichia coli alkaline phosphatase would dephosphorylate the synthetically prepared Coe-adenosylcobalamin 5'-phosphate, i.e. the physiological form, more readily than the cyano form.

It was deduced from this result, from the effect of tempera-

ture on the absorption spectrum and from the shape of the Arrhenius plot of the enzymatic dephosphorylation of cyanocobalamin 5'-phosphate, that the phosphatase preferentially attacks the base-off form of the substance, presumably because here the nucleotide carrying the 5' phosphate group had swung somewhat away from the bulk of the molecule.

It is of interest that

in this case the Cog-adenosyl group has the special function of facilitating, via a trans-effect, a biosynthetic transformation.

Studies with

heated P_. shermanii indeed directly showed extensive heat sensitivity of

336 the transformati on of Cog-adenosylcobalamin 5'-phosphate to Cop-adenosylcobalamin.

Since we did not like the idea of working with labile enzymes,

we did not study the possibly specific enzyme responsible for the final step in B-|2 biosynthesis any further.

The enzymatic formation of B 1 2 5 ' -

phosphate by P_. shermanii extracts has been studied by Renz (41-43). Cobalamin 5'-phosphate was shown to participate not only in the de novo biosynthesis of cobalamin, but also in a much less studied phenomenon, a base-exchange reaction, related to guided biosynthesis.

I t i s known (15,

44) that when a free benzimidazole i s administered to a bacterium containing a complete corrinoid such as for example incorporated while the adenine i s expelled.

2»

the benzimidazole i s

This reaction, however, does

not consist of a direct replacement of one base by another.

Renz has

shown that in the course of this exchange the original ribose i s removed along with the original base (45).

I t hence appeared possible that the

exchange entailed the participation of the N-a-glycosidic 5'-nucleotide of the new base.

When this hypothesis was tested i t was indeed found in

my laboratory that cobalamin 5'-phosphate i s an intermediate in the in vivo transformation (46).

No in v i t r o experiments on this reaction have

been performed so far.

The U t i l i z a t i o n

o f L - T h r e o n i n e as P r e c u r s o r

of

(R)-1-Amino-2-

Propanol The remaining part of the nucleotide loop i s the (R)-l-amino-2-propanol moiety.

I t has been known since the pioneering study of Krasna, Rosenblum

and Sprinson (47) that this i s derived from L-threonine. This work was 15 done with the N-labeled amino acid. Similar conclusions were obtained by Muller and Miiller (48) and by Lowe and Turner (49) with threonine.

14

C-labeled

Stereochemically the base corresponds to the amino acid minus

the carboxyl group.

One could readily conclude that we are here dealing

with a t r i v i a l problem, since i t should not be d i f f i c u l t to decarboxylate threonine enzymatically and to attach the resulting base in peptide l i n k age to the f propionic acid side chain on ring D of the corrin ring system.

However, no threonine decarboxylase has ever been reported.

On the

337 other hand it was known that synthetically prepared corrinoid containing threonine or O-phosphothreonine at the £ propionic acid group is not changed in vivo (50).

An alternative path for the formation of (R)-l-

amino-2-propanol from L-threonine had been proposed, namely a three-step sequence entailing enzymatic dehydrogenation of L-threonine to a-amino-Bketobutyrate, spontaneous decarboxylation to aminoacetone and its stereospecific reduction to (R)-l-amino-2-propanol (51).

The result of this

dehydrogenation, decarboxylation, reduction sequence is of course the net decarboxylation of the threonine.

This pathway by-passes the lack of a de-

carboxylase by utilizing the spontaneous decarboxylation of a e-keto acid. Both of the necessary dehydrogenases are known (51-59).

However, this

sequence, studied for example in E. coli (57) which does not produce B ^ » has never been shown in P_. shermanii grown under conditions where it produces the vitamin (57), and free (R)-l-amino-2-propanol does not appear to be a precursor in Streptomyces olivaceus (49) although contrary observations were reported with P_. shermanii (60).

In the course of studies on this

problem in my laboratory L.M. Cagen discovered a novel enzyme in £_. shermanii that uses inorganic pyrophosphate but not ATP to phosphorylate both L-threonine and L-serine (61, 62).

This enzyme, the only one known speci-

fically to phosphorylate the free amino acids, again had nothing to do with B-j2 biosynthesis, since it turned out to be absent from many organisms that do make corrinoids. It appeared necessary to invoke yet another pathway.

Work with Susan H.

Ford has, by a somewhat roundabout way, led to the proposal of a novel threonine-utilizing reaction sequence.

The first series of experiments

performed in my laboratory on this topic resulted in the partial purification of an enzyme system that catalyzes the reaction between L-threonine and Cog-adenosylcobyric acid to yield Cos-adenosylcobinamide (63).

Cobyric

acid is hexaamidated cobyrinic acid, i.e. the corrinoid fully amidated except for the (R)-l-amino-2-propanol group linked to propionic acid side chain f_.

In a crude £_. Shermanii system it was not possible to use L-

[U-^C]threonine, commercially available, for this study since it gave rise to a host of products which obscured the formation of cobinamide. Hence the source of radioactive marker was reversed, i.e. commercially unavailable ^C-labeled cobyric acid had to be incubated with unlabeled L-

338 threonine.

The labeled cobyric acid was prepared by two different methods,

either directly in the Coe-adenosyl form by isolation from

shermanii

that had been grown in the presence of [4-^C]6 -aminolevul inic acid, or by chemical breakdown of [^-C]cyanocobalamin isolated from £. shermanii. In the former case the Coe-adenosylcobyric acid was fractionated on Dowex 50 columns.

In the latter case the breakdown was accomplished both by the

zinc chloride-methanol method via the B-aminoisopropyl ester (43, 48), and by acid hydrolysis of [^ 4 -C]cobinamide (64), the main product. cyanocobyric acid obtained was converted to the

The labeled

[^-C]Co6-adenosylcobyric

acid by reaction in the dark with 5'-iodo-5'-deoxyadenosine, after reduction of the corrinoid with sodium borohydride (65-68). For the ensuing enzyme assays the cobinamide formed was distinguished from cobyric acid by ionophoresis at pH 6.5.

Purification by means of ammonium

sulfate fractionation after protamine sulfate treatment, followed by molecular sieving on a Bio-Gel P-6 column, effectively removed interfering enzymes that had prevented the use of labeled L-threonine with crude enzyme extracts.

Hence one could use parallel experiments with the purified sys-

tem to establish that cobyric acid was transformed to cobinamide. One 14 could start either with C-labeled cobyric acid and unlabeled L-threonine, 14 or with unlabeled cobyric acid and C-labeled L-threonine. Cobyric acid was active only in the CoS-adenosyl form.

Since the system required ATP

but no pyridine nucleotide, oxidized or reduced, two conclusions could be drawn: the ATP presumably energized the formation of the peptide bond between the amino group of the (R)-l-amino-2-propanol and the propionic carboxyl group, and aminoacetone was not an intermediate. Enzyme systems are, most of the time, smarter than the investigators, so that the in vitro system that carried out the over-all reaction did not by itself contribute more to the elucidation of its mechanism.

At this point

control experiments without enzyme revealed that threonine could in fact be decarboxylated, provided that both corrinoid and a reducing agent were present (69).

A reductant had always been added to the enzyme prepara-

tions, and their activity was lessened if this was omitted (63).

Since

we had here for the first time a non-enzymatic system for the decarboxylation of threonine, and since it required precisely some of the substances

339

that participate in the enzyme system, this partial system was investigated in some more detail.

It was found that the conditions for threonine decar-

boxylation mimicked the enzymatic ones quite closely.

Thus when diaquo-

cobyric acid was present, more rapid and more extensive decarboxylation occurred than when cyanoaquocobyric acid was used, and this, again, was better than cyanocobalamin.

Again, threonine and serine were decarboxyla-

ted much more extensively than other amino acids tried.

Furthermore,

6-alanine was not decarboxylated.

Hence an a-amino acid with a 6-hydroxyl

group was preferred for activity.

In terms of mechanism it could be shown

by means of chromatography on a Sephadex G-10 column that, in the presence of a reductant, an adduct was formed between corrinoid and threonine (or a threonine metabolite), since radioactive material was eluted earlier than threonine, in a region corresponding to the elution of the corrinoid (70). It was considered possible that some kind of ternary complex between threonine, corrinoid and sulfhydryl compound might be formed.

However, when

35 2-[

S]mercaptoethanol was used in the molecular sieving experiment, it was

found that no label was eluted in the region corresponding to corrinoid. Hence the reductant was not a part of the adduct formed.

Furthermore, the

system was unspecific for the type of reductant that could be used: in the presence of a non-thiol reductant such as sodium borohydride the adduct was also formed, and in fact CO^ was released more rapidly than with 2-mercaptoethanol.

Hence the role of the reductant was almost certainly to form

the B-|2r derivative (71, 72).

It was found, furthermore, that when the

absorption spectrum of a mixture of corrinoid, reductant and threonine in Tris-HCl buffer, pH 8, was compared to that of the mixture without threonine, a shift was detected that has been associated with Co-ligand formation (69).

The reaction was not limited to simple threonine decarboxyla-

tion, or to decarboxylations followed in part by some other chemical changes, since ionophoresis of the material that had been eluted from the Sephadex column in the threonine region revealed, in addition to (R)-lamino-2-propanol, a number of acidic substances.

The (R)-l-amino-2-propan-

ol was characterized as the dinitrophenyl derivative, as well as by thin layer chromatography on silicic acid, and by chromatography on paraffintreated and on acetylated paper (70). It was concluded that we here dealt with a model system for the first part

3k0

of the enzymatic reaction, and that one of the functions of the system

is to funnel the reaction in the direction of the

decarboxylation of L-threonine to (R)-l-amino-2-propanol.

enzyme

specific The spectral and

the molecular sieving results make it appear very likely indeed that threonine decarboxylation is a consequence of the attachment of threonine as a ligand to the corrinoid cobalt, and that in a subsequent step, not carried out by the model system, an inter- or intramolecular shift of the decarboxylation product to the f propionic acid group occurs. the adduct suggests a new intermediate in B ^

The formation of

biosynthesis, although this

point has not yet been tested with the enzyme system.

Acknowledgments Most of the author's work reported here was supported by Grant AM-09134 from the National Institutes of Health, United States Public Health Service.

The author is indebted for the help and collaboration received

over the years from the late Dr. Daniel L. Harris, in whose laboratory this work was started, two former graduate students, James A. Fyfe and Lauren M. Cagen, three post-doctorals, Karola Ohlenroth, Zenon Schneider and Susan H. Ford, and one former colleague, Charles L. Coulter.

References 1. Friedmann, H.C., Cagen, L.M.:

Ann. Rev. Microbiol. 24, 159-208 (1970).

2. Friedmann, H.C.: In B.M. Babior (ed.), Cobalamin - Biochemistry and Pathophysiology, John Wiley & Sons, New York, 1975, pp. 75-109. 3. Mervyn, L., Smith, E.L.:

Progr. Ind. Microbiol. 5, 152-201

4. Ford, J.E., Porter, J.W.G.:

Biochem. J. 51_, v (1952).

5. Ford, J.E., Holdsworth, E.S., Kon, S.K., Porter, J.W.G.: 150-151 (1953). 6. Ford, J.E., Holdsworth, E.S., Kon, S.K.: 7. Pawejkiewicz, J.:

(1964).

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9. DiMarco, A., Boretti, G., Ghione, M., Migliacci, A., Sanfilippo, A.: Ital. J. Biochem. (in English) 6, 259-269 (1957).

3 it 1

10. B a r c h i e l l i , R., B o r e t t i , G., J u l i t a , P., M i g l i a c c i , A . , Minghetti, A.: Biochim. Biophys. Acta 25, 452 (1957). 11. DiMarco, A . , B o r e t t i , G., M i g l i a c c i , A . , J u l i t a , P., Minghetti, A.: B o l l . Soc. I t a l . Sper. 33, 1513-1516 (1957). 12. B a r c h i e l l i , R., B o r e t t i , G., DiMarco, A., J u l i t a , A . , M i g l i a c c i , A . , Minghetti, A . , S p a l l a , C. : Biochem. J. 74, 382-387 (1960). 13. Pawejkiewicz, J . , Walerych, W., B a r t o s i r l s k i , B. : 6, 431-440 (1959).

Acta Biochim. Polon.

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15. Bernhauer, K., Becher, E . , Wilharm, G. : 248-258 (1959).

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16. B a r b i e r i , P., B o r e t t i , G., DiMarco, A . , M i g l i a c c i , A., S p a l l a , C.: Biochim. Biophys. Acta 57, 599-600 (1962). 17. DiMarco, A . , A l b e r t i , C.G., B o r e t t i , G., Ghione, M., M i g l i a c c i , A., S p a l l a , C. : Jjl H.C. Heinrich ( e d . ) , Vitamin B 1 2 and I n t r i n s i c Factor, 1st European Symposium, Hamburg 1956, Ferdinand Enke, S t u t t g a r t , 1957, pp. 55-59. 18. DiMarco, A . , B o r e t t i , G., S p a l l a , C.: ( i n English) 1 , 355-367 (1961). 19. B a r t o s i r l s k i , B.: (1966).

S c i . Rep. 1st Super Sanità

B u l l . Acad. Polon. S c i . , Ser. S c i . B i o l . 1£, 143-147

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22. Friedmann, H.C. H a r r i s , D.L.:

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24. Fyfe, J . A . , Friedmann, H.C.: 25. Takagi, Y . , Horecker, B . L . :

J. B i o l . Chem. 244, 1659-1666 (1969) J. B i o l . Chem. 225, 77-86 (1957).

26. Wright, R . S . , Tener, G.M., Khorana, H.G.: 2004-2006 (1958). 27. Friedmann, H.C., Fyfe, J . A . : 28. Hodgkin, D.C.:

J. B i o l . Chem. 244, 1667-1671 (1969).

Biochem. Soc. Symp. 1^3, 28 (1955).

29. F r i e d r i c h , W., Bernhauer, K.: 30. Friedmann, H.C.:

J. Am. Chem. Soc. 80,

Chem. Ber. 89, 2507-2512 (1956).

Methods Enzymol. 18B, 192-197 (1971).

31. Friedmann, H.C., G a r s t k i , C.: 54-58 (1973).

Biochem. Biophys. Res. Commun. 50

32. B o r e t t i , G., DiMarco, A . , Fuoco, L . , Marnatti, M.P., M i g l i a c c i , A . , S p a l l a , C.: Biochim. Biophys. Acta 37, 379-380 (1960). 33. Friedmann, H.C.: (1967).

Am. Chem. Soc., 154th Meeting, Abstract No. 109

342

34. Friedmann, H . C . :

J . B i o l . Chem. 243, 2065-2075

35. Friedmann, H . C . :

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36. Wagner, F . :

Biochem. Z. 336, 99-101

(1968).

(1971).

(1962).

37. C o u l t e r , C . L . , Hawkinson, S.W., Friedmann, H . C . : Biochim. B i o p h y s . Acta 177, 293-302 ( 1 9 6 9 ) . 38. Friedmann, H . C . :

Methods Enzymol. 18C, 92-95

(1971).

39. Hawkinson, S.W., C o u l t e r , C . L . , Greaves, M . L . : S e r . A, 318, 143-167 ( 1 9 7 0 ) . 40. S c h n e i d e r , Z . , Friedmann, H . C . : (1972).

Proc. Roy. Soc.

Arch. Biochem. B i o p h y s . 152, 488-495

41. Renz, P . :

Angew. Chem. ( I n t . Ed.) 6, 368-369

42. Renz, P . :

Hoppe S e y l e r ' s Z. P h y s i o l . Chem. 349, 979-981

43. Renz, P . :

Methods Enzymol. 18C, 82-92

44. Ford, J . E . , P o r t e r , J.W.G.: 45. Renz, P . :

London,

(1967). (1968).

(1971).

B r i t . J . Nutr. 7, 326-337

(1953).

Angew. Chem. ( I n t . Ed.) 4 , 527 ( 1 9 6 5 ) .

46. O h l e n r o t h , K . , Friedmann, H . C . : (1968).

Biochim. B i o p h y s . Acta 170, 465-467

47. K r a s n a , A . J . , Rosenblum, C., S p r i n s o n , D . B . : 745- 750 ( 1 9 5 7 ) . 48. M U l l e r , G., M ü l l e r , 0 . :

J . B i o l . Chem. 225,

Z. N a t u r f o r s c h . 21b, 1159-1164

49. Lowe, D . A . , T u r n e r , J . M . :

(1966).

J . Gen. M i c r o b i o l . 64, 119-122

(1970).

50. Bernhauer, K. , Wagner, F . :

Biochem. Z. 335., 325-339

51. Neuberger, A . , T a i t , G.H.:

Biochim. B i o p h y s . Acta 41, 164-165 ( 1 9 6 0 ) .

52. E l l i o t t , W.H.:

Biochem. J . 74, 478-485

53. Neuberger, A . , T a i t , G.H.:

(1962).

(1960).

Biochem. J . 84, 317-328 ( 1 9 6 2 ) .

54. T u r n e r , J . M . :

Biochem. J . 99^, 427-433 ( 1 9 6 6 ) .

55. T u r n e r , J . M . :

Biochem. J. 1 0 4 , 112-121

(1967).

56. Lowe, D . A . , T u r n e r , J . M . :

Biochim. B i o p h y s . Acta U O , 455-456

57. Lowe, D . A . , T u r n e r , J . M . :

J . Gen. M i c r o b i o l . 63, 49-61

58. Dekker, E . E . , Swain, R . R . : (1968).

Biochim. B i o p h y s . Acta 1_58, 306-307

59. Campbell, R . L . , Dekker, E . E . : 432-438 ( 1 9 7 3 ) .

Biochem. B i o p h y s . Res. Commun. 53,

60. M ü l l e r , G . , G r o s s , R . , S i e b k e , G.: 352, 1720-1722 ( 1 9 7 1 ) . 61. Cagen, L . M . , Friedmann, H . C . : 528-533 ( 1 9 6 8 ) . 62. Cagen. L . M . , Friedmann, H . C . : 63. Ford, S . H . , Friedmann, H.C.:

(1968).

(1970).

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Biochem. B i o p h y s . Res. Commun. 33.* J . B i o l . Chem. 247, 3382-3392

(1972).

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343

(1976). 64. Bonnett, R., Godfrey, J.M., Redman, D.G.: (1969).

J. Chem. Soc. (C), 1163

65. Murakami, M., Takahashi, K., Iwamoto, H., Matsumoto, J.: (in Japanese) 34, 598 (1966). 66. Yamada, R., Shimizu, S., Fukui, S.: 67. Hogenkamp, H.P.C. : 68. Dolphin, D.:

Bitamin

Biochemistry ]_, 1713-1719 (1968).

Biochemistry, 13_, 2736-2740

(1974).

Methods Enzymol. 18C, p. 41 (1971).

69. Ford, S.H., Friedmann, H.C.: 1083 (1976).

Biochem. ßiophys. Res. Commun. 72, 1077-

70. Ford, S.H., Friedmann, H.C.: (1977)1

Biochim. Biophys. Acta 500, 217-222

71. Peel, J.L.:

Biochem. J. 88, 296-308 (1963).

72. Pratt, J.M.: Inorganic Chemistry of Vitamin B 1 ? , Academic Press, New York, 1972, pp. 198-200.

R I B O S O M A L

P R O T E I N S

S H A R E

IN

VITAMIN

B

BIOSYNTHESIS

W.

Walerych

and

E.

Pezacka

D e p a r t m e n t of B i o c h e m i s t r y , 60-637 Poznan, Poland

Agriculture

University,

Introduction

O n e o f t h e t e r m i n a l s t a g e s of V i t . B ^

b i o s y n t h e s i s is the sequence

o f r e a c t i o n s l e a d i n g t o t h e i n c o r p o r a t i o n o f t h e N - oC - g l y c o s i d i c side into cobinamide.

A h y p o t h e t i c c o u r s e of those r e a c t i o n s

devised in Bernhauer's laboratory / l / . the b i o s y n t h e s i s of v i t a m i n B ^

Cobinamide

was

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

has a c o u r s e l i k e this:

cobinamide-P vitamin

nucleo-

> cobinamide-P-R

B ^

W h e a t m a d e i t p o s s i b l e f o r t h e a u t h o r s to f r a m e t h e h y p o t h e s i s w a s that they had i s o l a t e d a number of c o r r i n o i d compounds f r o m n a t u r a l sources. C.

Cobinamide phosphate d e r i v a t i v e s were isolated also f r o m

d i p h t h e r i a e and P.

s h e r m a n i i b y P a w e t k i e w i c z et a l .

/2,3/.

It w a s t h e r e s e a r c h w o r k o f t h e I t a l i a n g r o u p o f D i M a r c o e t that shed m o r e l i g h t on the p r o b l e m of t h i s s t a g e of v i t a m i n B synthesis.

al./4/ I

bio-

F r o m c u l t u r e of one of N o c a r d i a r u g o s a mutants the

a u t h o r s i s o l a t e d l a r g e r amounts of a compound in s t r u c t u r e v e r y much

© 1979 Walter de Gruyter & Co., Berlin • New York Vitamin BI2

3U6

like cobinamide ester guanosinediphosphate.

On a d d i n g 5, 6 - d i m e t h y l b e n z i -

m i d a z o l e to t h e b a c t e r i a l m e d i u m , v i t a m i n B ^ cobinamide.

The structural

a p p e a r d in place of

r e s e m b l a n c e o f G D P - c o b i n a m i d e to o t h e r

d e r i v a t i v e s of n u c l e o s i d e diphosphate i n v o l v e d reactions,

in other

biosynthetic

l i k e U D P - g l u c o s e i n the s y n t h e s i s of c a r b o h y d r a t e s o r

c h o l i n e in that of p h o s p h o l i p i d e s ,

of B ^

nucleoside and r e s u l t s in v i t a m i n B ^

plus GMP.

benzimidazole

The very

+

ATP +

»-

cobinamide-P

GTP

tions in vivo / 5 / .

+

GDP-cobinamide

T h i s h y p o t h e s i s was c o n f i r m e d by the

ADP +

and on

PP

Italian group in their

investiga-

They found a mutant of N o c a r d i a rugosa which did

not p r o d u c e v i t a m i n B ^ »

'3lJt

w a s

capable

d e r i v a t i v e s of the c o b i n a m i d e to v i t a m i n

c o n v e r t i n g the phosphate When biosynthesis

run on media containing radioactive phosphate, . When GDP-cobinamide was

added

it r e s u l t e d in

to t h e m

medium,

12

resulting

GDP-cobin-

in the r e a c t i o n as f o l l o w s :

cobinamide-P

ve B .

to

biosynthesis.

A c c o r d i n g to i t the G D P - c o b i n a m i d e d i r e c t l y r e a c t s w i t h

Cobinamide

CDP-

a l l o w e d the I t a l i a n i n v e s t i g a t o r s

f r a m e a n o t h e r h y p o t h e s i s e x p l a n i n g the mechanism

amide a r i s e s

GDP-

was

radioactithe

'

vitamin B ^

s h o w e d onl y i n s i g n i f i c a n t r a d i o a c t i v i t y .

l e d D i M a r c o a n d c o w o r k e r s to t h e a s s u m p t i o n t h a t t h e w h o l e phosphate molecule,

o r the c o b i n a m i d e phosphate f r o m

w e r e i n c o r p o r a t e d into vitamin B ^ 1. c o b i n a m i d e ,

2. c o b i n a m i d e - P ,

as p r e c u r s o r s of v i t a m i n B

anc

cobinamide

GDP-cobinamide,

' hence such compounds

3. G D P - c o b i n a m i d e ,

This

can be

as: regarded

biosynthesis.

In t h e i r o t h e r e x p e r i m e n t s the same i n v e s t i g a t o r s d e m o n s t r a t e d that D B I - n u c l e o s i d e as such was i n c o r p o r a t e d into v i t a m i n B ^ F r i e d m a n n et a l ,

/ 6 , 7, 8 / i s o l a t e d f r o m P . s h e r m a n i i

molecule.

-ribazole

which

3i»7

i s f o r m e d f r o m D B I a n d n i c o t i n i c a c i d m o n o n u c l e o t i d e i n the p r e s e n c e of t r a n s - N - g l y c o s i d a s e a n d p h o s p h a t a s e .

T h i s r e a c t i o n c a n be p r e -

sented as follows:

DBI

+

I ^ M N

oC - n b a z o l e - 5

P

ot - r i b a z o l e - 5 1 3 + nicotinate Phosphatase ., , _ oL - r i b a z o l e + P

T h e i n v i t r o i n v e s t i g a t i o n s of p a r t i c u l a r s t a g e s of v i t . B

1 ¿t

+

H

+

formation

f r o m c o b i n a m i d e h a v e not b e e n g i v e n a d e t a i l e d e l a b o r a t i o n s o f a r . In 1967, R o n z i o an B a r k e r r e p o r t e d a n e n z y m a t i c s y n t h e s i s of

GDP-

c o b i n a m i d e w i t h c o e n z y m a t i c f r o m of c o b i n a m i d e p h o s p h a t e a n d G T P / 9 / . T h e r e a c t i o n w a s p e r f o r m e d w i t h eel I - f r e e e x t r a c t f r o m P . s h e r m a n i i i n the p r e s e n c e o f r e d u c e d g l u t a t h i o n e .

T h e a u t h o r s f a i l e d to

d e m o n s t r a t e a n e n z y m a t i c s y n t h e s i s of c o b i n a m i d e p h o s p h a t e . substrate employed in their experiments w a s a chemically c o e n z y m a t i c f o r m of c o b i n a m i d e p h o s p h a t e .

The

synthesied

It w a s o n l y i n 1 9 6 8 that

R e n z /1 0 / c a r r i e d out a n e n z y m a t i c s y n t h e s i s of c o b i n a m i d e p h o s p h a t e . I n c o n t r a d i s t i n c t i o n to the d a t a b y R o n z i o a n d B a r k e r , o u t that c r u d e e x t r a c t s from

R e n z pointed

P . s h e r m a n i i o r the f r a c t i o n i s o l a t e d f r o m

them, b e i n g p r e c i p i t a d t e d with 4 0 - 6 0 % ammonium s u l p h a t e ,

catalyze

the s y n t h e s i s o f c o b i n a m i d e p h o s p h a t e f r o m c o b i n a m i d e a n d A T P .

The

a u t h o r a l s o p r e s e n t s a n i m p o r t a n t o b s e r v a t i o n that the c r u d e e x t r a c t s c a t a l y s e the f o r m a t i o n of v i t a m i n B I z- 5 p h o s p h a t e f r o m G D P - c o b i n a m i d e i n p r e s e n c e of D B I .

B u t a n a d d i t i o n of G T P to the r e a c t i o n

m i x t u r e r e s u l t e d i n the f o r m a t i o n of v i t a m i n B ^ of v i t a m i n B ^

ar|

d only small

amounts

phosphate.

T h e e x p e r i m e n t s c a r r i e d out i n o u r L a b o r a t o r y w e r e some i n t h e s t u d i e s o n the e n z y m a t i c s y n t h e s i s of v i t a m i n

progress VValerych,

K a t o a n d P a w e f k i e w i c z / l l / r e p o r t e d i n a b r i e f n o t e that E s c h e r i c h i a

3i+B

coli

1 1 3 - 3 mutant c o n t a i n s an e n z y m a t i c s y s t e m w h i c h c o n v e r t s

c o b i n a m i d e to v i t a m i n B ^ .

GDP-

T h e c o n v e r s i o n o c c u r s i n the p r e s e n c e of

D B I a n d N A D a n d , b e s i d e s the e n z y m e s of the p o s t r i b o s o m a l

fraction,

the r i b o s o m a l f r a c t i o n i s i n d i s p e n s a b l e f o r t h i s r e a c t i o n / 1 2 / .

The

s e q u e n c e s of these r e a c t i o n s c a n be p r e s e n t e d a s f o l l o w s :

NAD

»- N M N

+

_ . DMBIA

trans-N-gl ycosidase —

oC - r i b a z o l e - 5 ' P -ribazole-5'

P

... „ Vit. B

+ 12

adenosyl-GDP-cobinamide

_, „ -5 P

phosphatase. —

B e c k et a l . / 1 3 , 14, 1 5 / s h o w e d that

»-Vit. B

. . vitamin

- 5 ' P

„ B,„ 12

t h e i s o l a t e d r i b o s o m e s o f the

s p e c i e s Lactobacillus attach vitamin B

.

r e a c t

'

o n

of

converting

r i b o n u c l e o t i d e s into d e o x y r i b o n u c l e o t i d e s with c o e n z y m e B

taking

part i s dependent on vitamin B

But

and r i b o s o m e s being joined.

s t i l l i n s u f f i c i e n t l y e l u c i d a t e d a n d at t h e s a m e t i m e e x t r e m e l y i s the r o l e of r i b o s o m e s i n the p r o c e s s of v i t a m i n B

interesting

biosynthesis.

W e h a v e b e e n u s e d to t h i n k i n g that r i b o s o m e s a r e m e r e l y i n v o l v e d i n the p r o c e s s of p r o t e i n

biosynthesis.

R e s u l ts

T h e i n v e s t i g a t i o n s a i m e d at e l u c i d a t i n g t h e r o l e o f r i b o s o m a l in vitamin B

b i o s y n t h e s i s w e r e p e r f o r m e d on E s c h e r i c h i a coli

mutant i n c a p a b l e of s y n t h e s i z i n g c o r r i n o i d s . vitamin B

proteins

B e i n g a u x o t r o p h i c to

and cultured on vitamin-free media,

endogenous cobalamin.

1 13-3

the s t r a i n h a d no

F o r this s t r a i n a map of r i b o s o m a l p r o t e i n s

e l a b o r a t e d a n d c o m p a r e d w i t h t h o s e of the w i l d s t r a i n s / 1 6 / .

The

was

3U9

c o m p a r i s o n r e v e a l e d that the m u t a t i o n a l changes of the r i b o s o m e s involved in B ^

b i o s y n t h e s i s a r e of l i t t l e i m p o r t a n c e and m e r e l y

c o n c e r n the p r o t e i n s not d i r e c t l y i n v o l v e d i n the b i o s y n t h e s i s

studied.

T h e r e s u l t s obtained i n o u r L a b o r a t o r y s h o w e d that i n v i t a m i n B ^ synthesis are

a c t i v e both 7 0 S r i b o s o m e s f r o m E . c o l i and 5 0 S

bio-

subunits,

w h i l e 3 0 S s u b u n i t s remain inactive /table 1/.

T a b l e 1. A c t i v i t y of r i b o s o m a l s u b u n i t s i n v i t a m i n B

Ribosomes

% of G D P - c o b i n a m i d e ted to v i t a m i n

70S

20

50S

25

30S

-

biosynthesis

conver-

B ^

T h e h i g h e r a c t i v i t y of 5 0 S s u b u n i t s may be due to the fact that the r i b o s o m a l p r o t e i n s a c t i v e i n b i o s y n t h e s i s of the v i t a m i n a r e m o r e exposed

i n the s t r u c t u r e of 5 0 S s u b u n i t s than i n the p a r t i c l e s of 7 0 S

r i b o s o m e s / 1 7 , 18, 19/.

T h e r i b o s o m e s and t h e i r s u b u n i t s i s o l a t e d by d i f f e r e n t i a l f r o m the mutated s t r a i n ase a c t i v i t y /table 2 / .

of E . c o l i

centrifugation

1 1 3 - 3 w e r e f o u n d to have h i g h

RN-

350

T a b l e 2. R N - a s e a c t i v i t y in p a r t i c u l a r ribosomal p r e p a r a t i o n s

R N - a s e activity l/mg protein

Fraction Ribosomes 7 0 S

300, 0

F r a c t i o n S - 0 / a f t e r treating with / N H 4 / S 0 4 and centrifugatlon/

5,0

Fraction S - R

100,0

F r a c t i o n after Sephadex G - 5 0 fi 1 t r a t i o n P e a k 1

100,0

P e a k II

0,0

Peak III /S-50/

1,0

F r a c t i o n after Sephadex G - 2 0 0 P e a k II S - 2 0 0

0,0

F r a c t i o n s after Sephadex G - 1 0 0 P e a k II S - 1 0 0

0,0

T h e e x t i n c t i o n r a t i o s studied / O D 2 6 0 / 2 8 0 nm = 1. 89 OD 2 6 0 / 2 3 7 nm = 1 . 3 -

1. 9 5 and

1. 4 5 / a r e i n d i c a t i v e of obtaining " p u r e " r i b o s o -

mes f r e e of m R N A and c y t o p l a z m i c p r o t e i n s / 2 0 / .

In i s o l a t i o n the r i b o -

somal p r o t e i n s a c t i v e in the b i o s y n t h e s i s of cobalamin this high a c t i v i t y of endogenous ribosomal R N - a s e was put to good account. through d i a l y s i s the c o n c e n t r a t i o n of magnesium

Lowering

ions to 0. 4 mM would

induce removal of the i n h i b i t o r of R N - a s e and unwind the s t r u c t u r e of ribosomes / 2 l / .

In t u r n , an addition of ammonium sulphate up to 10 p e r

cent of s a t u r a t i o n of the system would inhibit the R N - a s e a c t i v i t y / 2 2 / and simultaneously salt out some f r a c t i o n of ribosomal p r o t e i n s .

Owing

351

T o the o p t i m a l c o n d i t i o n s b e i n g c r e a t e t f o r the a c t i v i t y of e n d o g e n o u s RN-ase

/23/,

nucleoproteid fragments were obtained /fraction

S-R/.

T h i s f r a c t i o n c a t a l y z e s the s y n t h e s i s of 6 0 0 pmol B ^ / m g of p r o t e i n / h . A n e l e c t r o p h o r e t i c i d e n t i f i c a t i o n of the r i b o s o m a l p r o t e i n s o b t a i n e d i n f r a c t i o n S - R p r o v e d a r e m o v a l of 19 b a s i c p r o t e i n s a n d o n e a c i d p r o t e i n of a l a r g e s u b u n i t out o f 5 0 S s u b u n i t s . S-R

The ribonucleoproteid

fraction

w a s f i r s t f i l t r e d o n gel S e p h a d e x G - 5 0 / F i g . 1 / .

FRACTION NUMBER

F i g . 1. E l u t i o n p a t t e r n f r o m S e p h a d e x G - 5 0 c o l u m n of r i b o s o m a l protein from 70S

ribosomes

It w a s o n l y the p r o t e i n p e a k I I I that d e m o n s t r a t e d a c t i v i t y i n c o b a l a m i n biosynthesis. Subsequently, /fig. 2/.

T h e a c t i v i t y w a s 2 5 0 0 0 - 4 0 0 0 0 p m o l / m g of p r o t e i n . this peak w a s r e c h r o m a t o g r a p h e d on S e p h a d e x

G-200

352

FRACTION NUMBER

Fig. 2. Elution pattern from Sephadex G-200 column of S-50 fraction

I n t h i s c a s e , p e a k II / S - 2 0 0 / d e m o n s t r a t e d s p e c i f i c a c t i v i t y of 5 0 0 0 0 pmol B

/ m g of p r o t e i n .

tion S - 2 0 0 ,

I n o r d e r to r e m o v e r R N A f r a g m e n t s f r o m f r a c -

the m e t h o d d e v i s e d by W i t t m a n et a l .

p r o t e i n s f r o m the r i b o s o m a l c o r e w a s e m p l o y e d .

/ 1 9 , 24/ for

isolating

T h e ribosomal

f r e e of r R N A f r a g m e n t s w e r e t h e n f i l t e r e d on S e p h a d e x G - 1 0 0 .

proteins The

p r o f i l e of e l u t i o n f r o m S e p h a d e x G - 1 0 0 c o l u m n / F i g . 3 / s h o w e d t h e p r e s e n c e of m a n y f r a c t i o n s .

T h e t h i r d of them / S - 1 0 0 / d e m o n s t r a t e d t h e

s p e c i f i c a c t i v i t y of 7 0 0 0 0 pmol B

/ m g of p r o t e i n .

2 6 0 / 2 8 0 nm of t h i s a c t i v e f r a c t i o n i s l e s s t h a n o n e ,

The extinction ratio which is indicative

of t h e p r o t e i n p r e p a r a t i o n b e i n g d e p r i v e d of R N A / 2 4 / .

A

two-dimensio-

nal e l e c t r o p h o r e s i s of the f r a c t i o n o n p o l y a c r y l a m i d e gel r e v e a l e d of five basic proteins / F i g . 4 / . T h r e e of t h e m ,

L5,

L18,

o t h e r two, S 7 and S 1 0 ,

L 2 5 s p e c i f i c a l l y bind with 5 S R N A , with 3 0 S s u b u n i t s / 1 9 / .

a n d the

Those proteins are

353

l 80

1

1 o - r i b a z o l e - 5 P „ 12

+

P. i

+

ADP +

PP

5'AMP

oL - r i b a z o l e - 5 1 = L18 protein

357

„ _, „ 6. v i t a m i n B ^ S P

—

phosphatase -

„ vitamin B ^

Reactions 4 and 5 a r e closely connected with ribosomes.

The

+

„ P.

results

o b t a i n e d s e e m to be i n d i c a t i v e of a n e w r o l e of r i b o s o m e s i n the s y n t h e s i s of b i o l o g i c a l a c t i v e n o n p r o t e i n c o m p o u n d s .

A c k n o wl e d g e m e n t s

W e a r e to g r a t e f u l to P r o f e s s o r J e r z y P a w e t k i e w i c z f o r h e l p f u l

discus-

sions and his v a l u a b l e a d v i c e .

References

1. D e l l w e g ,

H. , B e c h e r ,

E.,

Bernhauer,

K . : B i o c h e m . J.

327 . 422

/1956/. 2. P a w e t k i e w i c z , 3. P a w e t k i e w i c z , P o l o n . _6_, 4 3 1 4. Di M a r c o , A . , 5. B a r b i e r i ,

J.,

Zodrow,

K. : A c t a M i c r o b i o l .

J,,

Walerych,

W. , Bartosinski,

P o l o n . _6_, 2 1 9 / 1 9 5 7 / B. : Acta Biochim.

/1959/. Spalla,

C . : G i o r n e l e M i c r o b . _9_, 2 3 7 / 1 9 6 1 / .

P . , B o r e t t i , G. , Di Marco, A. , Migliacci,

A. , S p a l l a , C . :

B i o c h i m . B i o p h y s . A c t a 57_, 5 9 9 / 1 9 6 2 / . 6. F r i e d m a n n ,

H . C . : J. B i o l . C h e m .

7. F r i e d m a n n ,

H. C. , F y f e ,

8. F r i e d m a n n ,

H. C. , Cagan,

243,

2065

/1968/.

J. A . : J. B i o l . C h e m .

244 , 1667 / 1 9 6 9 / .

L . M . : A n n . R e v . M i c r o b i o l o g y 24 ,

159

/1970/. 9. R o n z i o ,

R . A . , B a r k e r , H . A . : B i o c h e m i s t r y _6_, 2 3 4 4 / 1 9 6 7 / .

10. R e n z P . : B i o c h e m . B i o p h y s . R e s . C o m m . _30, 11. W a l e r y c h , Comm.

T. , Pawetkiewicz,

J. ; B i o c h e m . B i o p h y s . R e s .

3J_, 3 2 8 / 1 9 6 8 / .

12. W a l e r y c h , 13. B e c k ,

W. , Kato,

373,/1968/.

W . : R o c z n i k i W S R - P r a c e h a b i I i t a c y j n e 1_6_, / 1 9 6 8 / .

W. S. , Hardy,

J. : P r o c . N a t l . A c a d . S c i . _54, 2 8 6 / 1 9 6 5 / .

358 14. K a s h k e t ,

S. , Beck,

W. : Biochem. Z .

15. K a s h k e t ,

S. , Kaufman,

J. T . , B e c k ,

342 , 449 / 1 9 6 5 / . W. ; Biochim. Biophys. Acta 64,

447 / 1 9 6 2 / . 16. P e z a c k a - F o j u d z k a , 17.

Issinger , O . G . ,

E. , Walerych,

W. : F E B S L e t t e r s 65, 99 / 1 9 7 6 /

K i e f e r , M. C . , T r a u t , R. R. : E u r . J. Biochem.

59,

137 / 1 9 7 5 / . 18. L i t m a n n , D . J . , C a n t o r , C . R . : B i o c h e m i s t r y J_3, 3 / 1 9 7 4 / . 19. W i t t m a n , 20. 21.

H . G . : E u r . J. B i o c h e m . _6J, 1 / 1 9 7 6 / .

S p i t n i k - E I son, P . ,

A t s m o n , A . : J. M o l . B i o l .

Petermann, M. L. : " T h e Physiol, Ribosomes". New York

and Chem.

4 5 , 113 / 1 9 6 9 / . P r o p e r t i e s of

ed. E l s e v i e r P u b l i s h i n g Company.

Amsterdam-London-

/1974/.

22. S c h m i d t , G . : In T h e E n z y m e s ed.

Boyer,

P . D . , Academic

Press,

N e w Y o r k - L o n d o n , _5_, 3 7 / 1 9 6 1 / . 23.

Williams,

24.

Wittmann-Liebold, 571

F . R. : B i c h i m . Biophys. A c t a 147, 4 2 7 / 1 9 6 7 / .

25. E r d m a n n , 26. D o v g a s ,

V . A . , N o m u r a , M. : P r o c . Nat. A c a d . S e i . N. V. , Markova,

Alakhov,

33^

Hörne,

30. G r u m m t ,

Kaplan, /1955/.

T . A . , V i n o k u r o v , L . M.,

R. A. , Le B r e t , M. , L e P e c q ,

R. , G a r r e t t ,

R. ,

Stöffler,Q:

133 / 1 9 7 3 / .

J. R . , E r d m a n n , F . , Grummt,

31. K o r n b e r g ,

12 / 1 9 7 1 / .

93, 535 / I 9 7 5 / .

P. N. , Bellemare, G. , Monier,

J. M o l . B i o l . _77_,

68,

lu. A . : F E B S L e t t e r s 5 3 , 3 / 1 9 7 5 / .

J. , M o n i e r , R . , G a r r e t ,

I. B . : J . M o l . B i o l . 28. G r a y .

L. F . , Mednikova,

lu. B . , O v c i n n i k o v ,

27. F e u n t e u n ,

29.

B. , Wittmann, H. G. : Biochim. Biophys. A c t a

/1974/.

V. A. : Proc. Nat. Acad. Sei.

I. : F E B S L e t t e r s 4 2 ,

A . : In Methods in E n z y m o l o g y ,

O. N. : A c a d e m i c P r e s s .

ed.

70,

10 / 1 9 7 3 / .

3 /1974/. Colowick,

Inc. P u b l . N e w Y o r k ,

S. P. ,

vol.

II

122

THE RIBOSOMALS PROTEINS L2, L5, L18 AND L25 INVOLVED IN VITAMIN B BIOSYNTHESIS. E. Pezacka and W. Walerych I n s t i t u t of B i o c h e m i s t r y , A g r i c u l t u r e U n i v e r s i t y , 60-637 Poznan, Poland.

The b i o s y n t h e s i s of vitamin B ^

i s probably a m u l t i s t e p r e a c t i o n catalyzed

by a c o n s i d e r a b l e number of enzymes. The g r e a t e r part of these enzymes

is

unknown. Though some of the intermediate products are known the exact s e quence of r e a c t i o n s i s s t i l l

under d i s c u s s i o n . One of these r e a c t i o n s

the i n c o r p o r a t i o n of the 5,6-dimethylbenzimidazolyl corrinoid.

is

nucleotide into the

In t h i s r e a c t i o n GDP in GDP-cobinamide i s s u b s t i t u t e d by benz-

imidazolyl nucleotide c a l l e d ct-ribazol or a - r i b a z o l - 5 ' - p h o s p h a t e

(1,2).

I t was found that at t h i s r e a c t i o n stage of the s y n t h e s i s of vitamin in E. c o l i , f r e e system r e q u i r e s the presence of ribosomes

B^

(3,4,5,6).

Results The ribosomes were i s o l a t e d from E. c o l i 113-3 s t r a i n by p u r i f y i n g them through d i f f e r e n t i a t i n g c e n t r i f u g a t i o n in presence of 0.5 M NH^Cl. By the c r e a t i o n of optimal c o n d i t i o n s f o r the ribosomal RN-ase r e a c t i o n a c t i v e RNA-proteins complex was l i b e r a t e d from ribosomes ( 7 ) . Further

purifica-

t i o n of t h i s a c t i v e ribosomal complex was c a r r i e d out on Sephadex G-50, and G-200 columns employing b u f f e r s of d i f f e r e n t i o n i c

strength.

The p r o t e i n s from these s p e c i f i c complex were i d e n t i f i e d by two-dimensional gel e l e c t r o p h o r e s i s as a c i d ribosomal p r o t e i n s L5, L18, L25 and S1, S10. At the f i n a l

stage of p u r i f i c a t i o n a c t i v e p r o t e i n s were obtained by

preparative two dimensional gel e l e c t r o p h o r e s i s . The a p p l i c a t i o n of t h i s method enabled both d e f i n i t e i d e n t i f i c a t i o n of ribosomal p r o t e i n s i n vitamin

involved

b i o s y n t h e s i s and e v a l u a t i o n of i n t e r r e a c t i o n s between these

proteins. In this reaction proteins s p e c i f i c a l l y

i n c i d e n t to 5S RNA i . e .

L5, L18 and L25 are d i r e c t l y i n v o l v e d . I t concerns protein L2 as w e l l , however i t s s i t e in ribosome s t r u c t u r e i s so f a r unknown ( 8 , 9 ) .

© 1979 Walter de Gruyter & Co., Berlin • New York Vitamin Bu

3GD

Jh rQ IT

V t

- f t - i

i i ' ' i i i i i i i i i i i i i i i i i i i i i i i i i i i i i ii 12 3 4 5 6 7 8 9 0 12 K * « » O S » » 3 0 32 34 NUMBER OF RIBOSOMAl PROTEINS

I t was stated that in vitamin

b i o s y n t h e s i s reaction protein L18 s p l i t s

the diphosphate bond in GDP-cobinamide and not in NAD molecule or in another ribose donor. Protein L2 stimulates the e f f i c i e n c y of t h i s

reaction.

The other proteins i . e . L5 and L25 p o s i t i v e l y influence the s t a b i l i z a t i o n of enzymatic a c t i v i t y of i s o l a t e d ribosomal proteins. I t i s very important that chloramphenicol, vernamycin and f u s i d i c acid (but not erythromycin) were potent i n h i b i t o r s of the reaction vitamin B ^ b i o s y n t h e s i s . These a n t i b i o t i c s are well-known as i n h i b i t o r s of the protein b i o s y n t h e s i s . Furthermore i t was discovered that in s u b s t i t u t i o n reaction of GDP into benzimidazolyl nucleotide in GDP-cobinamide, ribosomes i s o l a t e d from wild s t r a i n s of E. c o l i

can a l s o take part.

References 1. Friedmann, H.C., J.Biol.Chem., 243, 2065 (1968) 2. Friedmann, H.C., in "Methods in Enzymology", Vol. 18C. Acad. P r e s s , New York, p.92, 1971. 3. Walerych, W., Kato, T. and Pawelkiewicz, J. , B.B.R.Commun., 3 U 3 (1968). 4. Pezacka, E . , Walerych, W. and Pawelkiewicz, J . , in Abstracts of the IX FEBS Meeting, Budapest, 1974, No. 387. 5. Pezacka, E. and Walerych, W., FEBS Letters 65, 99 (1976). 6. Pezacka, E. and Walerych, W., in Abstracts of the X I I FEBS Meeting, Dresden, 1977. 7. Petermann, M.L., in "The P h y s i o l o g i c a l and Chemical Properties of Ribosomes". Ed. E l s e v i e r Publishing Co., Amsterdam, 1974. 8. Wittmann, H.G., Eur.J.Biochem., 61_, 1 (1976). 9. Erdmann, V.A., S p r i n z l , M. and Richter, D. and Lorenz,S., Acta B i o l . Med. Germ. 33, 605 (1974).

FORMATION AND ROLE OF VITAMIN B12 IN PROTAMINOBA CTER RUBER AND RIIIZOBIUM MELILOTI.

K. Sato, S. Inukai, S. Ueda, T. Seki and S. Shimizu Department of Food Science and Technology, Nagoya University Chikusa-ku, Nagoya 464, Japan

Introduction Methanol-utilizing bacteria have received much attention as a potential source for producing single cell protein (SCP), while nitrogen-fixing bacteria have become the center of wide interest for exploitation of nitrogen fixation with a low expence of energy. to form cobalamins (1, 2, 3, 4, 5). role of cobalamins in these bacteria.

Both bacteria are known

However, there are few studies on the Elucidation of the role of

cobalamins in the cells will help not only understanding the physiology of these bacteria, but also extending practical applications.

Furthermore,

it may be useful as a model system for understanding the function of cobalamins in higher organisms. The present report aims at presenting basic data on the role of cobalamins in Protaminobaeter ruber NR-1, a methanol-utilizing bacterium and Rhizobium meliloti 1480, a symbiotic nitrogen-fixing bacterium.

A.

Results on P. ruber

1)

Forms of cobalamins in P. ruber.

P. ruber NR-1 isolated by us (4, 6)

has similar features to Pseudomonas AM-1, which produces a considerable amount of cobalamins (5) and also forms bacteriochlorophyll under certain cultural conditions in spite of being classified as nonphotosynthetic bacteria (7).

The bacterium was cultivated in a medium containing

inorganic salts and methanol as a sole carbon and energy source (4, 6, 7).

© 1979 Walter de Gruyter & Co., Berlin • New York Vitamin B»

362

The amount of cobalamin produced by P. ruber was about 150 to 200 ng per ml of culture broth and almost all of cobalamins remained in the cells (4). In order to determine the forms of cobalamins in the cells, cobalamin compounds were extracted by boiling with ethanol and purified with phenol extraction and the column chromatography on DEAE-cellulose, Amberlite XAD-2, P-cellulose and Sephadex G-25 (4). the dim light.

All the operations were carried out in

At the stage of P-cellulose column chromatography,

cobalamin compounds were separated into two species.

They were identified

as methylcobalamin and adenosylcobalamin by various tests. The base moiety was identified as 5,6-dimethylbenzimidazole according to the method of Barker et at. (8).

The species of the upper ligand was

determined by whether cobalamin compounds could act as the coenzyme of methylcobalamin : homocysteine methyltransferase (4) or dioldehydrase (dl-1,2-propanediol hydro-lyase EC 4.2.1.28) (9). 2)

Time course of the forms of cobalamin compounds and cobalamin-dependent

enzyme systems.

Time course of the forms of cobalamins during the

cultivation revealed that more MeCbl existed in the cells of earlier phases of growth and that almost all of cobalamins existed in the form of AdoCbl in the stationary phase (4).

We also found two cobalamin-dependent

enzyme systems in P. ruber: that is, methionine synthetase in which MeCbl participates (4) and methylmalonyl-CoA mutase in which AdoCbl participates (10, 11).

Figure 1 shows the time course of cell growth and the specific

activities of the both enzymes during the growth.

The patterns of these

activities corresponded well to the contents of MeCbl and AdoCbl in the cells.

The specific activity of methylmalonyl-CoA mutase was comparable to

that in Propionibacterium shermanii known as the best source of methylmalonyl-CoA mutase (12).

Since the bacterium contained the enzymes

catalyzing the reactions from propionyl-CoA to succinyl-CoA and from a-ketoglutarate to a-hydroxyglutarate, we tentatively proposed a metabolic sequence as shown in Fig. 2 (11).

3)

Interconversion of methylcobalamin and adenosylcobalamin.

In order to

detect the changes of cobalamin-forms and enzymatic activities in a short period of cultivation, we adopted the following cultivation method (13). The stationary phase cells cultivated with ["^Co]CN-Cbl were harvested

363

Cultivation time (h) Fig. 1 Time course of the bacterial growth and the specific activities of methylmalonyl-CoA mutase and methionine synthetase. Methylmalonyl-CoA mutase and N^-methyltetrahydrofolate: homocysteine methyltransferase were assayed according to the methods of (11) and (4), respectively.

Tricarboxylic acid cycle

r GoA

t

Glyoxylate

Malate

Î Fumarate

t

I Isocitrate

Acetyl-Ccrt Propionyl-CoA j^COî

«-Hydroxyglutarate

Methylmalonyl-COA

\

j, (Adenosyl-B« ) — Succinyl-CoA *

\

cc-Ketoglutarate

Succinate Fig. 2 Proposed metabolic sequence leading to the formation of succinate via a-hydroxyglutarate in P. ruber.

36k

aseptically and suspended in a fresh, sterilized and cobalt-deficient medium of 2-fold volume. in 500 ml-flask

Each 100 ml-suspension was aerobically cultivated

for the necessary period at 30 °C.

Cobalamins were

extracted from the cells with 80 % ethanol by boiling for 20 min.

Carrier

cobalamins were added to the ethanol extract and the sample was purified with phenol extract or Amberlite XAD-2 (50 ^ 100 mesh) column ( 0 . 6 x 3 . 0 cm) chromatography, in which cobalamins adsorbed were washed with deionized water and eluted with 25 % ethanol (13).

The partially purified sample was

subjected to paper electrophoresis and found to consist of only MeCbl and AdoCbl.

The amount of each cobalamin on the radioactigraph was measured.

Cell-free extracts were prepared by grinding with alumina from the cells cultivated in a similar way with non-labelled cobalamin.

Figure 3 shows

the time course of the changes of cobalamin-forms and cobalamin-dependent methionine synthetase.

It was confirmed that, although almost all of the

cobalamins had existed as AdoCbl in the inoculum cells, MeCbl and methionine synthetase were formed in the early phase of the bacterial growth and very rapidly decreased after the late logarithmic phase.

This

suggests that there is an interconversion between MeCbl and AdoCbl.

The

conversion of MeCbl into AdoCbl was demonstrated in the cell-free system of P. ruber.

ATP, homocysteine, and FMNH2 as well as MeCbl were indispensable

z Cultivation

time

(h)

Fig. 3 Time course of the bacterial growth, the changes of cobalaminforms and the specific activity of methionine synthetase.

365 for the AdoCbl formation (13).

Therefore, we supposed the following

reaction scheme. Reducing system AdoCbl

MeCbl Homocysteine 4)

ATP

Methionine

PPPi

Catalytic sites of cobalamin-dependent methionine synthetase.

Figure 4

shows time course of the specific activities of N5 -methyltetrahydrofolate : homocysteine methyltransferase and methylcobalamin : homocysteine methyltransferase.

In comparison with the rapid decrease of the former

activity, the latter one did not decrease so much.

Would this imply that

both reactions are catalyzed by the different enzymes?

In the case of

Escherichia coti, both enzymatic reactions are known to occur at separate sites on the same enzyme (14, 15).

Therefore, cobalamin-dependent

methionine synthetase from P. ruber was purified nearly to homogeniety with affinity chromatography on a cobalamin-Sepharose (16).

Both enzymatic

activities were detected in the purified enzyme as demonstrated in E. coli 8!

%ISin

Ur

n

g w10

Î1*

2.1 oj 2 g

fi 2 9

i1 £ Ol Z

2

20

40

60

Cultivation

80

100

time

(h)

120

140

Fig. 4 Time course of the bacterial growth and the specific activities of N^-methyltetrahydrofolate: homocysteine methyltransferase and MeCbl: homocysteine methyltransferase.

366 and the purification degree based on each methyltransferase activity was almost similar (17).

The results shown in Fig. 3 may be explained by

assuming that the active site of N^-methyltetrahydrofolate : homocysteine methyltransferase is inactivated more rapidly than the active site of methylcobalamin : homocysteine methyltransferase.

The remaining activity

of the latter might facilitate the decomposition of methylcobalamin released from the active site of the former, i.e. N5 -methyltetrahydrofolate : homocysteine methyltransferase as the inactivation would proceed. No detection of OH-Cbl and sulfitocobalamin in the cells of P. ruber suggests that there is little possibility of the existence of (S-Co)Cblenzyme, which is an inactive form of methionine synthetase proposed in E. ooli (15, 18). The conversion of AdoCbl to MeCbl in vitro remains to be demonstrated.

It

is unknown yet whether P. ruber has an enzyme resembling to "holoenzyme synthetase" obtained from E. ooli, which catalyzed the reaction of apoenzyme with AdoCbl to form holoenzyme in the presence of a reduced pyridine nucleotide (19), but at least some enzymatic activation step of cobalamin into MeCbl would exist in the cells of early phase of the growth (Fig. 3).

MeCbl formation and high specific activity of methionine

synthetase in the early phase probably imply the important roles of this enzyme system in the cell multiplication.

B.

Results on R. meliloti.

1)

Effect of various additions to cobalt*~deficient medium on the growth of

R. meliloti.

Evans et al. found in their pioneering work that leguminous

plants grown under symbiotic conditions required cobalt ion and contained cobalamin in the nodules and that the cobalt ion requirement and cobalamin formation were due to the bacteria in the nodules (20).

Furthermore, they

found cobalamin-dependent enzyme systems, methylmalonyl-CoA mutase (21) and ribonucleotide reductase (22, 23) in R. meliloti.

However, they were

unable to find any growth factor for R. meliloti by examining whether the additions such as nucleosides to strictly cobalt-deficient medium could maintain the bacterial growth in place of cobalt ion (23).

As shown in

367

Fig. 5, we found that the addition of methionine as well as cobalamin to the medium could replace cobalt ion required for the growth of B. meliloti.

Other compounds

such as nucleosides and succinate were not effective at all (24, 25).

We also

could demonstrate the cobalamindependent methionine synthetase in the cell-free extract of the bacterium (24).

These

findings led to the question of how deoxyribonucleotide would be supplied in the cells cultured in a cobalt-deficient, methionine-supplemented medium. Table 1 shows the levels of methionine synthetase and ribonucleotide reductase from the cells cultured with cobalt ion or with methionine in the absence of cobalt ion. Under the latter conditions, the level of methionine synthetase was very low, while that of ribonucleotide reductase was relatively high

20

30

AO

50

Cultivation time(hr) Fig. 5 Effect of various additions to the cobalt-deficient medium on the growth of R. meliloti. (O—O), cobalt ion, 1.0 ppb; ( • — • ) , OH-Cbl, 20 ppb; (• • ), methionine, 0.5 mM or 1.0 mM; (A A), methionine, 0.1 mM; (A A ) , methionine, 0.05 mM; (X X), none (or succinate, 370 yM; glutamate, 670 yM; deoxyribose, 50 yM and adenine, guanine, uracil and cytosine, each at 12.5 yM; deoxyadenosine, deoxyguanosine, deoxycytidine and thymidine, each at 40 yM or 400 yM).

and a large amount of apo-ribonucleotide reductase was inducibly formed. At least, the critical level of the reductase needed for cell multiplication seems to be maintained by the trap of a trace amount of cobalamin, which was produced from a very little contaminated cobalt ion with the abundunt apoenzyme.

3GB

Table 1

Levels of cobalamin-dependent methionine synthetase and ribonucleotide reductase in R. meliloti

Additions to the cobaltdeficient medium

1480.

Specific activities (nmoles/mg/h) Methionine synthetase Ribonucleotide reductase - AdoCbl + AdoCbl - MeCbl + MeCbl

Cobalt ion (50 ppb) Methionine (0.5 mH)

56.9

58.0

4.8

0.3

3.7

1.7

4.1 823*

The reaction Mixtures for the assay of methionine synthetase contained n5-[14C] methyltetrahydrofolate, SO moles; homocysteine, 500 moles; SAM, 25 moles; DTT, 500 naoles; FMNH2> 200 moles; potassium phosphate buffer, 25 Maoles; MeCbl, 5 moles and cell-free extract in a total volume of 0.5 al. The incubation was for 30 ain at 30°C in the dark under hydrogen gas. The mixtures for the assay of reductase contained [14C]C0P, 50 naoles; ATP, 0.1 {moles; magnesium chloride, 1.5 yaoles; DTT, 10.0 joules; potassium phosphate buffer 25 males; AdoCbl 10.0 moles and cell-free extract in a total volume of 0.5 ml. Incubation was for 60 ain at 37*C in the dark. *

2)

Incubation period was for 5 min.

Purification and properties of ribonucleotide reductase.

Apo-

ribonucleotide reductase from the cells cultivated in the cobalt-deficient, methionine-supplemented medium was purified by the purification steps summarized in Table 2. Table 2

The purified enzyme showed a single band in agar

Purification of ribonucleotide reductase from fihizobium meliloti

Step

Volume

Protein

Total activity

ml

wtq

units*

A. Crude extract

245

4,974

10,270

B. Streptomycin

320

C. Ammonium sulfate

—

66

1,307

D. 1st DEAE-cellulose

104

E. 2nd DEAE-cellulose

35

8,700

Specific activity

Yield

units/mg 2.1 —

« 100 85

7,120

5.5

69

258

8,670

33.6

84

99

4,990

50.4

49

F. Sepharose 6B fraction No. 14

9

9.0

670

74.1

No. 15

9

12.1

940

77.9

No. 16

9

11.0

830

75.4

No. 17

9

8.1

600

73.8

* One unit of activity corresponds to the formation of 1 ymole of dADP from ADP per hour.

30

369

gel electrophoresis and in isoelectric focusing.

Furthermore, it had a

single symmetrical peak with S2o>w of approximately 20 at pH 9.0 in sedimentation velocity determination. concentration at pH 7.5.

The enzyme aggregated in high

As shown in Table 3 (26, 27, 28), the molecular

weight of the ribonucleotide reductase was aa. 500,000 (without aggregation), but nearly 1,000,000 in an aggregated form at pH 7.5.

From

the behavior of SDS polyacrylamide gel electrophoresis, the enzyme having the molecular weight of 500,000 was assumed to consist of three subunits of molecular weight of 130,000 and one subunit of molecular weight of 110,000.

This differs completely from ribonucleotide reductase of L.

leiohnannii, which is shown to be composed of a single polypeptide chain having a molecular weight of 76,000 (29, 30).

Most remarkable feature of

Rhizobium reductase is that the substrate is nucleoside diphosphate instead of nucleoside triphosphate required by L. leichmcmnii enzyme.

In R.

meliloti, was rather complicated than that of L. leiohmannii, and dGTP and dCTP had more diverse effect as shown in Fig. 6 (31). Table 3

Comparison of the properties of ribonucleotide reductase from the different organisms E.

coli27) L.

leichmannii^S)

R.

meliloti2g)

Substrate

CDP

CTP

CDP

Hydrogen transport system

NADPH —»(SH)2

NADPH — • (SH)2 —•> AdoCbl[H]

Metal requirement

Mg2+

NADPH — * (SH) 2 —»AdoCbl[H] ( M g 2+)*

Activator

ATP

dATP

dATP

Single polypeptide

3Si + S 2

Inhibitor

ATP

Subunits

Bl + B 2

Molecular weight

Bi 160,000 B2 78,000 aa.240,000

Optimal pH *

•v 7.5

Conditional requirement

(Mg 2+ )*

51 52 76,000

7.5 -x. 8.0

130,000 110,000

aa. 500,000 (pH 9.0) aa.1,000,000 . ( PH 7.5 aggregated 9.0 % 10.0

370

Control of in

deoxyribonucleotide R.meliloti

formation

1480

ADP

GDP

dADP

dATP dCDP*

*••••• dCTP

CDP

UDP positive effect Fig. 6

weak positive effet

Control of deoxyribonucleotide formation in R. meliloti

1480.

» 3)

Morphological change of R. meliloti 1480.

The addition of methionine

to the cobalt-deficient medium almost restored the bacterial growth, but the elongate form (5 ^ 7 U) of the bacterium did not change to normal size (1 ^ 2 u) under such conditions (25).

For the conversion of the cells to

normal size, it was necessary to add cobalt ion with methionine. Since folic acid with methionine did not have any effect for the growth (32) and the level of methionine synthetase was restored to the normal one when the cells became normal size by adding cobalt ion with methionine, the interpretation of cell elongation

by the trap hypothesis of N5-methyl-

tetrahydrofolate seems to be difficult and cobalamin-dependent enzyme might have some important role which could not be replaced by mere addition of methionine.

The significance of cobalamin-dependent methionine synthetase

for cell multiplication would be stressed in relation to the existence of higher proportions of MeCbl in younger tissues and the highest proportion (40 %) in fetal tissues (33) which well corresponds to the maximum volue of MeCbl content in the bacterial cells of early growth phase (13, 34).

371 Acknowledgement

The authors are grateful to Prof. R. H. Abeles, Brandeis University, U. S. A. and Mr. E. Hiei

for the study on affinity chromatography of

cobalamin-dependent methionine synthetase in P. ruber and to Prof. R. L. Blakley, University of Iowa, U. S. A. for a generous gift of L. leiahnannii ribonucleotide reductase.

References

1. Tanaka, A., Ohya, Y., Shimizu, S., Fukui, S.: 921-924 (1974).

J. Ferment. Technol. 52,

2. Toraya, T., Yongsmith, B., Tanaka, A., Fukui, S.: 477-479 (1975). 3. Nishio, N., Yano, T., Kamikubo, T.: 207-213 (1975). 4. Sato, K., Ueda, S., Shimizu, S. : (1977).

Appl. Microbiol. 30,

Agric. Biol. Chem. 39, 21-27,

Appl. Environ. Microbiol. J33, 515-521

5. Kamikubo, T., Hayashi, M. , Nishio, N., Nagai, S.: Microbiol. 35, 971-973 (1978).

Appl. Environ.

6. Shimizu, S., Kobayashi, T., Sato, K., Ohmiya, K., Mori, M., Nishimura, T., Sasaki, M.: Nogei Kagaku Kaishi (in Japanese) 52, 477-484 (1978). 7. Sato, K.:

FEBS Lett. 85, 207-210 (1978).

8. Barker, H. A., Smyth, R. D., Weissbach, H., Toohey, J. I., Ladd, J. N., Volcani, B. E.: J. Biol. Chem. 235, 480-488 (1960). 9. Lee, H. A., Abeles, R. H.:

J. Biol. Chem. 238, 2367-2373 (1963).

10. Sato, K., Ueda, S., Shimizu, S.:

FEBS Lett. 71, 248-250 (1976).

11. Ueda, S., Sato, K., Shimizu, S.: (1978).

J. Nutr. Sei. Vitaminol. 24, 477-489

12. Barker, H. A.: The enzymes (3rd ed., Academic Press, New York and London) 6, 511-524 (1972). 13. Sato, K., Shimizu, S.: 14. Taylor, R. T.:

Unpublished results.

Arch. Biochem. Biophys. 144, 352-362 (1971).

15. Taylor, R. T., Weissbach, H.: The enzymes (3rd ed., Academic Press, New York and London) 9, 121-165 (1973). 16. Sato, K., Hiei, E., Shimizu, S., Abeles, R. H.: (1978). 17. Sato, K. , Hiei, E., Shimizu, S.:

FEBS Lett. 85, 73-76

Submitted to "Vitamin" (in Japanese).

372 18. Taylor, R. T., Weissbach, H.: (1969).

Arch. Biochem. Biophys. 129, 745-766

19. Brot, N., Weissbach, H.:

J. Biol. Chem. 241, 2023-2028 (1966).

20. Ahmed, S., Evans, H. J.: (1961).

Proc. Natl. Acad. Sci. U. S. 47, 24-36

21. DeHertogh, A. A., Mayeux, P. A., Evans, H. J.: 2446-2453 (1964). 22. Cowles, J. R., Evans, H. J.: (1968).

J. Biol. Chem. 239,

Arch. Biochem. Biophys. 127, 770-778

23. Cowles, J. R., Evans, H. J., Rüssel, S. A. : 1460-1465 (1969).

J. Bacteriol. 97_,

24. Sato, K., Inukai, S., Shimizu, S.: 723-728 (1974).

Biochem. Biophys. Res. Commun. 60,

25. Inukai, S., Sato, K. , Shimizu, S.: (1977).

Agric. Biol. Chem. 41, 2229-2234

26. Inukai, S., Sato, K. , Shimizu, S.: (No. 3) (1979).

Accepted in Agric. Biol. Chem. 44,

27. Thelander, L., Sjöberg, B. M., Erikson, S.: Methods in Enzymol. (Academic Press, New York, San Francisco and London) _5]_, 227-237 (1978). 28. Blakley, R. L.: Methods in Enzymol. (Academic Press, New York, San Francisco and London) 51, 246-259 (1978). 29. Panagou, D., Orr, M. D., Dunstone, J. R., Blakley, R. L.: 11, 2378-2388 (1972).

Biochemistry

30. Chen, A. K., Bhan, A., Hopper, S., Abrams, R., Franzen, J. S.: Biochemistry 13, 654-661 (1974). 31. Inukai, S., Sato, K. , Shimizu, S.:

Unpublished results.

32. Sato, K., Seki, T., Inukai, S., Shimizu, S.:

Unpublished results.

33. Linnell, J. C.: Cobalamin (ed. by Babior, B. M.) pp. 287-333 (Fig. 6-8), John Wiley & Sons (1975). 34. Sato, K., Shimizu, S., Fukui, S.: (1971).

Agric. Biol. Chem. 35, 333-337

CURRENT STATUS OF THE MECHANISM OF ACTION OF B

-COENZYME

R.H. Abeles Graduate Department of Biochemistry, Brandeis University Waltham, Massachusetts 02154

Dioldehydrase, an enzyme which requires a B ^ - c o e n z y m e , catalyzes the conversion of several diols to the corresponding aldehydes.

CH 3 -CHOH-CH 2 OH CH 2 OH-CH 2 OH

>

CH 3 -CH 2 -CHO

->

CH 2 0H-CH0H-CH 2 0H

CH 3 -CHO

>

CH 2 OH-CH 2 -CHO

CH 2 F-CH0H-CH 2 0H

CH 2 F-CH 2 -CHO

The work of Arigoni and his colleagues has established that the reaction proceeds with the intermediate formation of a 1,1'-gem-diol: CH 3 -CHOHCH 2 OH

—

CH3-CH2-C(OH)2H

>

CH 3 -CH 2 -CHO

The formation as well as the dehydration of the intermediate gem-diol is enzyme catalyzed.

The reaction catalyzed by dioldehydrase is, therefore,

analogous to other B ^ - c o e n z y m e dependent reactions and can be represented by the following scheme: I

I

I

I

X

H

H

X

In all Bj 2 -coenzyme dependent reactions, an interchange takes place between a group X and a hydrogen on an adjacent carbon.

© 1979 Walter de Gruyter & Co., Berlin • New York Vitamin Bu

nu, In Figure 1, a mechanism is shown for the reaction catalyzed by dioldehydrase.

According to this mechanism, a complex is formed between coenzyme

and apoenzyme. lytically.

In this complex, the carbon-cobalt bond is cleaved homo-

However, at this stage, only a small fraction of the coenzyme

E CH.R

r

1

[Co] SH

It

,

E + [Co]

_1_

.SH EC

.CH2R

E^T

CH B R [Co]

CHZR

E -

[Co]

PH

PH *CHTR

E

^[Co]

is present in the dissociated form.

J* [Co]

Upon combination with the substrate,

the equilibrium is shifted and approximately 60% of the coenzyme is converted to the dissociated form.

Dissociation of the coenzyme leads to

the formation of an adenosyl radical (-CH^R).

This radical abstracts a

hydrogen from the substrate (SH) to produce a substrate radical (S-) and 5'-deoxyadenosine (CH^R).

(S-) rearranges by an, as yet, unspecified

mechanism to (P•)• CH3

Ad

5'-deoxyadenosine

375 The coenzyme is then regenerated and the final product (PH) is formed through reversal of the events just described. We shall next consider various aspects of this mechanism and the supporting experimental evidence.

First, we shall consider the hydrogen transfer

process.

Hydrogen Transfer When the conversion of 1,2-propanediol to propionaldehyde is carried out 2 3 in

H^O or

H^O, no isotopic hydrogen is found in the reaction product.

The hydrogen, which is transferred from C-l to C-2, is transferred without exchange with solvent protons.

Dr. Zagalak carried out experiments de-

signed to determine whether this hydrogen transfer is intramolecular or intermolecular. In these experiments, he allowed the enzyme to act on a 3 mixture of 1- H, 1,2-propanediol and unlabeled ethylene glycol. He then isolated the products, acetaldehyde and propionaldehyde, and determined 3 3 their H content. He found that acetaldehyde containined H. When ethylene glycol is present in large excess over 1,2-propanediol, essentially all tritium in the product is present in acetaldehyde.

Therefore, hydrogen

transfer can be intermolecular, although some intramolecular transfer may occur.

This point will be considered later.

The occurrence of inter-

molecular hydrogen transfer indicates that at some intermediate stage in the reaction, there must be, somewhere on the enzyme, at least two equivalent hydrogens.

One of these is supplied by the substrate and the other(s)

by the enzyme-coenzyme complex. 3

These results further suggested that if 3

the enzyme acts on 1- H-propanediol, coenzyme complex.

H should be found in the enzyme-

This was confirmed experimentally.

It was also shown

that all of the tritium present in the enzyme-coenzyme complex is located at the C-5' position of the coenzyme.

376

Ad

H CH2-C-

Co

H CH

CH

OH

OH

Furthermore, it was shown that both hydrogens at the C-5' position, although stereochemically not equivalent, become labeled.

This observa-

tion suggested that at some stage during the reaction, C-5' loses its asymmetry.

This could most reasonably occur as a result of cleavage of

the C-Co bond. At this point, it was concluded that the coenzyme serves as intermediate 3 hydrogen carrier, i.e.,

H from C-l of the substrate is first transferred

to the coenzyme and from there, to the C-2 position of the product.

An

experiment was then carried out by P. Frey and M. Essenberg to determine whether the kinetics of tritium transfer are consistent with such a process. This experiment consisted of two parts: 1) Unlabeled 1,2-propanediol 3 was allowed to react with enzyme- H-C-5'-coenzyme and the rate of tritium 3 transfer from coenzyme to product was measured. 2) 1- H, 1,2-propanediol was added to enzyme-coenzyme (unlabeled).

Aliquots were removed periodi-

cally and the specific activity of the coenzyme and propionaldehyde were determined. These experiments allowed us to establish whether sufficient 3 3 H was present in the coenzyme at each time point so that the H of the product could be derived from the coenzyme.

It was found that the specific 3 activity of the coenzyme was sufficiently high so that all of the H in the

product could be derived from the coenzyme. A reaction pathway in which 3 H occurs entirely through the coenzyme is therefore permissable on kinetic grounds.

377 Some additional information was gained from these experiments:

It was

found that one ^H atom was transferred from coenzyme to product per 250-300 turnovers, i.e., the tritium isotope effect is 125-150 (assuming an intermediate with three equivalent hydrogens).

This is an unusually

large isotope effect! It was also found that during the course of the reaction, the specific activity of the coenzyme was 300-3000 fold higher than that of the sub3 strate. This observation shows that H cannot be introduced into the coenzyme by isotope exchange between enzyme bound reaction product and coenzyme. The results described so far, show that intermolecular hydrogen transfer occurs.

We next carried out an experiment to determine whether intra3 molecular transfers occur. 1- H-1,2-propanediol was added to dioldehydrase in the presence of unlabeled ethylene glycol.

A series of experiments

with increasing concentrations of ethylene glycol were carried out.

The

specific activity of propionaldehyde was determined as a function of ethylene glycol concentrations and extrapolated to infinite ethylene glycol concentration.

It was found that at infinite ethylene glycol con-

centrations, the specific activity of propionaldehyde reached a finite value.

From these results, it was calculated that intramolecular transfer

occurred in 1 out of 100 turnovers. The experiment just described indicates that the

3 H isotope effect is

approximately 50. It will be recalled that in a previous experiment, 3 H transfer from coenzyme to substrate was measured, an isotope

where

effect of 125-150 was found.

What is the reason for the discrepancy?

It is possible, but not likely, that this discrepancy represents experimental uncertainty.

The two numbers were determined through entirely

different experiments and involved extrapolations.

Alternatively, the

results suggest that tritium derived from the substrate has a somewhat better chance of transfer to the reaction product than tritium from the coenzyme.

This could possibly be due to restricted rotation of the

methyl group of 5'-deoxyadenosine, but other possible explanations can be

378 envisioned.

The results described so far are entirely consistent with the mechanism shown in Figure 1.

According to this mechanism, the hydrogen of the

substrate molecule becomes equivalent with the two hydrogens of the C-5' position of the coenzyme due to the formation of 5'-deoxyadenosine.

Con-

sequently, a product molecule will acquire either the hydrogen derived from its substrate precursor, or one of the hydrogens of the coenzyme. This is equivalent to saying that both inter- and intramolecular hydrogen transfer can occur.

Both of these processes have been demonstrated.

2 The mechanism of Figure 1 also predicts that with 1- H, 1,2-propanediol, 3 3 the probability of H transfer from H containing coenzyme to reaction 2 product should be increased. When a substrate containing H reacts with tritiated coenzyme, the methyl group of 5'-deoxyadenosine will contain 3 2 1 3 1 H, H, H. When a non-isotopic substrate reacts, it will contain H, H, 1 3 H. Due to isotope effects, the probability of transferring H to product 3 2 1 from a methyl group containing H, H, H, is higher than from a methyl 1 1H. The data in Table I shows that the probabilgroup containing 3H, H, 3 ity of finding H in the product is much higher when a deuterated substrate 3 is used. With a non-isotopic substrate, H transfer from coenzyme to product occurs in one out of 310 catalytic events, while with a deuterium containing substrate, it occurs in one out of 14 catalytic events.

Table I Rate of

Substrate

H Transfer from Coenzyme to Product

Rate of Product Formation

ta)

c h 2 o h CH OH C2H2OH C2H20H

11.8 sec" 1

3.6

3 Rate of H Transfer to Product (b) 0.038 sec

0.26

1

a/b

310

14

379

These experiments show that a substrate-derived hydrogen and at least one coenzyme-derived hydrogen compete for transfer to the reaction product. It does not necessarily mean that this occurs at every turnover, since the substrate derived hydrogen could be "stored" in the coenzyme.

A

further experiment was, therefore, carried out to establish that at every catalytic event, a selection is made from a hydrogen pool which contains 2 the substrate hydrogen. In this experiment, a mixture of 1- H,l,2-propanediol and unlabeled ethylene glycol was added to dioldehydrase and 3 C-5'- H-coenzyme.

The specific activity of propionaldehyde and acetalde-

hyde was determined.

The specific activity of propionaldehyde, i.e., the

product derived from the deuterated substrate, was higher than that of acetaldehyde.

When ethylene glycol was present in large excess over pro-

panediol, the specific activity of propionaldehyde was four times that of acetaldehyde.

When the deuterated substrate was in excess, the specific

activity of propionaldehyde was eight times that of acetaldehyde.

The

presence of deuterium in the substrate increases the probability of tritium * transfer to the reaction product.

In the experiments in which the un-

labeled substrate was present in excess, deuterium from the deuterated substrate could not be "stored" in the coenzyme.

Therefore, we concluded

that in all catalytic events, the substrate derived hydrogen is part of a hydrogen pool from which the product hydrogen is derived.

Intermediate Involvement of 51-Deoxyadenosine The data discussed so far are consistent with the intermediate involvement of 5'-deoxyadenosine as shown in Figure 1.

It is clearly desirable

to show the reversible formation of 5'-deoxyadenosine directly.

The first

evidence that the adenosyl moiety of the coenzyme could be converted to 5'-deoxyadenosine was obtained with substrate analogues such as glycolaldehyde and chloroacetaldehyde.

When these compounds were added to the

enzyme-coenzyme, a catalytically inactive complex was formed. *

When this

The same results were obtained when deuterated ethylene glycol and nonisotopic propanediol were used.

380 complex was denatured under a variety of conditions, 5'-deoxyadenosine was isolated.

The amount of 5'-deoxyadenosine formed was nearly equivalent to

the amount of enzyme-bound coenzyme. It is, of course, essential to demonstrate the formation of 5'-deoxyadenosine under conditions where no enzyme inactivation occurs.

We interrupted

the catalytic reaction under a variety of conditions, but were unable to detect significant amounts of 5'-deoxyadenosine although trace amounts were found.

The inability to detect significant quantities of 5'-deoxy-

adenosine may be due to the low steady state concentration of that compound. Evidence for the reversible formation of 5'-deoxyadenosine was obtained with ethanolamine deaminase.

This enzyme catalyzes the following reaction:

B12-Coenzyme CH -CH | 2 | 2 OH NH+

CH t -CH0

+

NH^ 4

The work with ethanolamine deaminase was carried out in collaboration with Professor B. Babior.

With this enzyme, as with dioldehydrase, only trace

amounts of 5'-deoxyadenosine can be detected when the catalytic process is interrupted.

2-NH2-propanol is a substrate for this enzyme, although

the rate of reaction is considerably slower than with the normal substrate It was found that when the catalytic process was interrupted in the presence of 2-NH2~propanol, by denaturing the enzyme with trichloroacetic acid, 5'-deoxyadenosine could be isolated.

Approximately 60% of the co-

enzyme was converted to 5'-deoxyadenosine.

5'-Deoxyadenosine was now iso-

lated from an enzyme-coenzyme complex which was catalytically active. Presumably, in the presence of 2-amino-propanol, the steady state concentration of 5'-deoxyadenosine is higher than in the presence of the normal substrate, ethanolamine.

To demonstrate the reversible formation of 3 5 -deoxyadenosine, the following experiment was carried out: 1- H, 2-NH21

propanol was added to enzyme-B 14 C

-coenzyme.

(The coenzyme was labeled with

in the adenosyl moiety to facilitate analysis.)

was divided into two portions.

The reaction mixture

One portion was denatured and the amount

381 of B^-coenzyme and 5'-deoxyadenosine were determined.

To the other

portion, ethanolamine was added and then the enzyme was denatured. coenzyme and 5'-deoxyadenosine were again determined.

Ethanolamine was

added to displace 2-NH2~propanol from the enzyme and thus reduced the steady state concentration of 5'-deoxyadenosine. experiment are summarized in Table II.

The results of this

When the enzyme-coenzyme

complex is denatured after addition of l- 3 H-2-NH 2 -propanol, 75-80% of the coenzyme is converted to 5'-deoxyadenosine.

Table II Reversible Formation of 5'-Deoxyadenosine

Isotope Exp A

Exp B

5-d-Ad

DBCC

Acetaldehyde

3

H

1.95 x 10 5 dpm

0.2 x 10 5 dpm

14

C

3.0 x 10 4

0.63 x 10 4

3

H

.07 x 10 5

.06 x 10 5

4 0.44 x 10

4 3.08 x 10

14

C

E-DBCC( 1 4 C-4.05 x 10 4 dpm)

+

1.82 x 10 5

1- 3 H, 2-NH 2 ~propanediol

A Denature

I 3 I 2 OH NH 3 V denature isolate DBCC, 5'-d-Ad CH 3 CH0

382 3

5'-Deoxyadenosine contains

H derived from the substrate.

When the complex

was denatured after addition of ethanolamine (the steady state concentration of 5'-deoxyadenosine is reduced), essentially no 5'-deoxyadenosine was found but B^-coenzyme was present.

Furthermore, all of the counts

originally present in 5'-deoxyadenosine are now found in acetaldehyde derived from unlabeled ethanolamine.

Clearly, 5'-deoxyadenosine was con-

verted to coenzyme after addition of ethanolamine.

These experiments

establish that 5'-deoxyadenosine can be formed reversibly.

Radical Intermediate The first conclusive evidence for radical formation was obtained by Professor B. Babior.

He showed that addition of substrate to ethanol-

amine deaminase-B^2 coenzyme gave rise to ESR signals. detected, one corresponded to Bj^fr) signal.

anc

* t'le

ot

^er

t0

an

Two signals were organic radical

We obtained similar results with dioldehydrase.

With isotopi-

cally labeled substrate analogs, it was shown that part of the organic radical is located on the substrate.

Under steady state conditions,

approximately 60% of the coenzyme is converted to

It was also

shown that the rate of formation of the radical species is sufficiently rapid to allow it to be an intermediate in the catalytic process. Professor Sands has analyzed the ESR spectrum obtained in the presence of o substrate and has concluded that the substrate radical is 7 A removed from the cobalt radical.

Therefore, covalent bond formation between sub-

strate and the cobalt of the coenzyme is unlikely. Since B i2(r)

can

react with

is inaccessible to

it is very likely that enzyme bound

B

j2(r

Together with Dr. Mildvan, we carried out NMR

experiments in which we attempted to measure the effect of the enzyme bound radical upon the water relaxation rate.

No effect was seen and it o

was concluded that no H^O molecule can approach to within 12 A of the cobalt atom.

383 In the mechanism of Figure 1, we have indicated that the carbon cobalt bond of the enzyme bound coenzyme is partially dissociated, although the equilibrium is far in favor of the undissociated form.

The enzyme-coenzyme com-

plex, in the absence of substrate, gives no ESR signal.

However, in the

absence of substrate, the enzyme bound coenzyme reacts with 0^. coenzyme does not react with 0^)• the coenzyme in some way.

(The free

The enzyme must, therefore, activate

A reasonable mechanism for this activation is

the homolytic dissociation of the C-Co bond. Additional evidence for the radical mechanism is provided by results from Professor Arigoni's laboratory.

He has examined the conversion of stereo-

specifically labeled ethanolamine to acetaldehyde and found that the resulting acetaldehyde was racemic.

D

H

H

NH 2 OH

D

chiral

0 "H

racemic

D NH 2 H

The most reasonable explanation for the racemization is the intermediate formation of a radical.

38ft

Role of the Apoprotein in the Catalytic Process

So far, in all discussions of the mechanism of action of B^-coenzymes, the role of the apoprotein has been completely ignored. obvious functions which the apoprotein must fulfill: ^12(r)' i-e-> prevent its reaction with 0^.

There are some

it must stabilize

It must prevent intermediate

species such as 5'-deoxyadenosine, the adenosyl radical and substrate and product radicals from escaping from the active site.

We, therefore,

tested the interaction of various proposed intermediates with dioldehydrase.

It was found that dioldehydrase binds Bj2(r) stoichiometrically

and essentially irreversibly. react with 0 T h e way:

B

^2(r) bound to dioldehydrase does not

binding experiment was carried out in the following

enzyme and B^(- r y generated by photolysis of B^-coenzyme, were in-

cubated for approximately 5 min under anaerobic conditions and then exposed to air.

The reaction mixture was then passed through a Bio-Gel P-6

column (1.2 x 11 cm) equilibrated with 0.05 M potassium phosphate buffer pH 8.0, 2% 1,2-propanediol.

The spectrum of the protein showed that

I?12(r) was present in nearly stoichiometric amounts.

The protein eluate

was dialyzed (aerobically) for twenty-four hours without significant dissociation or oxidation of B,„, ,. 12 (r) Next, the interaction of apoenzyme and 5'-deoxyadenosine was examined. No irreversible binding of 5'-deoxyadenosine was detected. However, when 14 the enzyme was incubated with C -5'-deoxyadenosine and then with ^ ^ ( r ) or B

. ,, followed by molecular exclusion chromatography as described - i4

1 ( a

above, C

-5'-deoxyadenosine was associated with the protein.

5'-Deoxy-

adenosine remained associated with the protein even after prolonged dialysis.

The amount of 5'-deoxyadenosine bound depended upon the con-

centration of 5'-deoxyadenosine in the original reaction mixture, but reached a maximum of one mole of 5'-deoxyadenosine per mole of enzyme, ^ass ^12(r)

5'-deoxyadenosine with the apoprotein is approximately 10"\ or

®12(a)

t0

a

When

P o e n z y m e prior to addition of 5'-deoxy-

adenosine, no 5'-deoxyadenosine is associated with the protein after Bio-Gel P-6 chromatography.

When

re

P-^ ace ^ by methyl-cobalamin,

5'-deoxyadenosine is not associated with the protein after Bio-Gel P-6

385 chromatography. The mechanism of Figure 1 requires that at an intermediate stage during the catalytic process, a state of the enzyme exists in which present and 5'-deoxyadenosine is very tightly bound. that such a state can be reconstituted.

is

We have now shown

It seems reasonable to assume

that 5'-deoxyadenosine is bound in a cleft of the enzyme.

When

B

j2(r)

binds, the cleft becomes blocked and the dissociation of 5'-deoxyadenosine is prevented.

Apparently when methyl-cobalamin binds to the enzyme, the

cleft does not become completely closed and 5'-deoxyadenosine can escape. Although one of the functions of the apoenzyme is the protection and stabilization of reactive intermediates, it seems very likely that the apoenzyme participates in other important ways in the catalytic process.

An

obvious function appears to be the promotion of the homolytic cleavage of the carbon-cobalt bond.

During a discussion of this topic some years

ago, Professor Eschenmoser suggested that this process might be facilitated by a distortion of the corrin ring brought about by the interaction between coenzyme and apoenzyme.

The corrin ring of the coenzyme has six

amide groups around its periphery.

The possibility occurred to us that

these amide groups might hydrogen bond to the carbonyl group of the protein and that this interaction may be important for the activation of the coenzyme.

To test this point, we prepared coenzyme analogs in which one

of the three propionamide groups was modified by replacing the -NH^ group with either -OH, -OCH^, or -NHCH^.

The reactivity of some of these

analogs is shown in Table III. Table III Reactive Rates of Coenzyme Analogs b site

-COO86

e site

-CONHCH, 41

-CONHCH3

-COOCH 3

16

14

-COO-

-COOCH

11 DBCC = 100

7

386 The results show that replacement of a propionamide group, even by another neutral group, can reduce the catalytic efficiency significantly.

In each

case, a reduction in the steady state concentration of the radical species corresponding to the reduction in catalytic efficiency was observed. The coenzyme analogs also differ from the normal coenzyme in other respects. The analogs bind reversibly to the apoenzyme, whereas the normal coenzyme binds essentially irreversibly.

The complex between several analogs and

the apoenzyme is not 0^ sensitive in the absence of substrate.

The analogs

which form an 0 2 stable complex with the apoenzyme are the analogs in which the e-propionamide group is replaced by -COO

or -NHCH^.

I have

previously mentioned that the complex between apoenzyme and the normal coenzyme is inactivated by 0^ in the absence of substrate.

A possible

explanation of this lack of 0^ sensitivity is that in the complex between coenzyme analog and apoenzyme, homolytic cleavage of the carbon-cobalt bond does not occur in the absence of substrate.

These results are consistent

with, but do not prove, the hypothesis that interaction between side chain amide groups and apoenzyme is important in bringing about the homolytic cleavage of the carbon-cobalt bond. The mechanism in Figure 1 is now supported by considerable experimental evidence.

I would like to emphasize that many of the key intermediates

^ 1 2 ( r ) ' ^'-deoxyadenosine) have been definitely identified and their reversible formation has been demonstrated.

Strong evidence exists also

for other proposed intermediates (various radical species).

Other mech-

anisms have been proposed which involve the intermediate formation of B

12(s)' k u t

n0

direct evidence exists for its formation during the cata-

lytic process.

Before closing, I would like to make a few remarks about other B^-coenzyme dependent reactions.

It is very likely that for the dioldehydrase and

ethanolamine deaminase, the conversion of Sa radical rearrangement.

P- (Figure 1) involves

This, however, is not necessarily true for

other B^-coenzyme dependent rearrangements.

These could proceed by car-

bonium or carbanion mechanisms as required by the particular reaction.

387 This is illustrated in Figure 2.

It is, for instance, possible, as shown

in Figure 2, that the initial substrate radical transfers an electron to B ^ C r ) to form a substrate derived carbonium ion and B j2(s)

CH2R

SH —> CH3R

PH CH2R

ZÌ7

^ "

388 The rearrangement would then occur through a carbonium ion mechanism. Similarly, carbanion mechanisms could occur.

It is one of the unique

properties of the corrin system that +1, + 2, +3 oxidation states of cobalt are approximately of the same stability.

This property may well be res-

ponsible for the versility of the coenzyme. The work which I have described here was carried out by the following people:

H.A. Lee, 0. Wagner, B. Zagalak, P.A. Frey, M. Essenberg, T. Carty,

J. Valinsky, E. Krodel, K. Sato, T. Toraya, and A. Cheung. cially supported by the National Institutes of Health.

It was finan-

A S T E R E O C H E M I C A L A P P R O A C H TO T H E D I O L D E H Y D R A T A S E

D.

REACTION

Arigoni

Eidgenössische Technische Hochschule Zürich, Laboratorium Organische Chemie, CH-8006 Zürich, Switzerland

The enzyme diol dehydratase talyzes the irreversible propionaldehyde and ethylene a s

enzyme

from Aerobacter aerogenes

glycol

in a similar way w i t h

a cofactor made

it p r o b a b l e

from the

s i m p l e t h a n s u g g e s t e d by a c u r s o r y and the subsequent

d e n c e for m a n y u n u s u a l

features

for

co-

beginning

w a s far

less

i n s p e c t i o n of t h e i r

studies have

into

glycerol

(1,2). The o b s e r v e d requirement

t h a t t h e m e c h a n i s m of t h e s e t r a n s f o r m a t i o n s

chiometry

ca-

c o n v e r s i o n of p r o p a n e - 1 , 2 - d i o l

and interacts

für

stoe-

indeed provided

i n t h e m o d e of a c t i o n of

evithis

e n z y m e . M o s t of t h e e x p e r i m e n t a l w o r k h a s f o c u s s e d o n t h e panediol-propionaldehyde (3 - 10) a r e A first

condensed

in Pig.

striking property

both enantiomers

r e a c t i o n and the relevant

of t h e e n z y m e

t h e m into the same achiral p r o d u c t . g e n a t o m f r o m C - l of t h e s u b s t r a t e

is t h a t

center

causing a net

it w i l l

as s u b s t r a t e s

and

In b o t h p r o c e s s e s is t r a n s f e r r e d

t r a n s i e n t l y m o d i f i e d f o r m of t h e c o e n z y m e

a t i o n at t h i s

accept

convert a hydro-

first to

(5,8) a n d t h e n

a

back

i n v e r s i o n of t h e

configur-

(3,10). D u r i n g the t r a n s f e r the

migrating

atom mixes with the hydrogen atoms of t h e m o d i f i e d c o f a c t o r

in the C-5' m e t h y l e n e

(9) a n d a l a r g e

isotope

feature of the enzyme resides

it) 1979 Walter de Gruyter & Co., Berlin • New York Vitamin B l2

group

effect,

k„/k = 10 - 1 2 , h a s b e e n o b s e r v e d f o r t h e o v e r a l l H D a t i o n (6). A second surprising

results

1.

of t h e p r o p a n e d i o l

t o C - 2 of t h e p r o d u c t

pro-

transform-

in the

un-

390

0H

u

H-i-i.0

„•

I -c(.HVC-H I CH,

-C-OH

ch3 ID)

& C - H HÌ-C-H

Y H-C-0

"

Figure 1

(R)

HO,

tL.,

X

(S) C

Y

"

«

H

Figure 2

(R)

=*•

HO-C-vOrH

V

I

(S)

• H,*0

I

j*2

^

ch 3

CH 3

HO—C-^OjH

.

Y

ch2

ch2

CH 3

CH3

Figure 3

*

HjO

S

39 1 usual differentiated stereospecificity

l i n k e d up w i t h the h y -

d r o g e n a b s t r a c t i o n step. W h e r e a s w i t h the

(R)-isomer as a s u b -

strate l a b i l i z a t i o n is r e s t r i c t e d to the H „ - a t o m at C - l , r e n a c t i o n of the (S)-isomer i n v o l v e s s p e c i f i c m i g r a t i o n of the Hg-atom

(7). T h u s , it seems that the e n z y m e can

differentiate

the d i a s t e r e o t o p i c H - l i g a n d s w i t h i n the m e t h y l e n e group of a g i v e n s u b s t r a t e only by f e e l i n g the c o n f i g u r a t i o n of the

ad-

j a c e n t chiral c e n t e r at C - 2 . A f u r t h e r a n d m o s t s i g n i f i c a n t facet of the r e a c t i o n w a s

re-

v e a l e d by the o b s e r v a t i o n that the o r i g i n of the o x y g e n a t o m in the a l d e h y d e g r o u p of the p r o d u c t v a r i e s w i t h the a t i o n of the s u b s t r a t e

(4). S t a r t i n g w i t h the

configur-

(S)-isomer

oxy-

g e n from C - l is r e t a i n e d in the f o r m a t i o n of the p r o d u c t , but w h e n the a l d e h y d e is g e n e r a t e d f r o m the

(R)-isomer its

oxygen

a t o m stems e n t i r e l y f r o m the C-2 h y d r o x y l group of the

pre-

cursor. Since a r e a r r a n g e m e n t of the c a r b o n s k e l e t o n has b e e n r u l e d out by a p p r o p r i a t e

l a b e l l i n g experiments s one m u s t

clude that at least in the case of ( R ) - p r o p a n e d i o l

con-

conver-

s i o n into the a l d e h y d e i n v o l v e s m i g r a t i o n of an h y d r o x y l f r o m C-l to C - 2 . The m o s t e c o n o m i c a l

group

i n t e r p r e t a t i o n of t h e s e

p u z z l i n g r e s u l t s r e s t s o n the r e a s o n a b l e a s s u m p t i o n of a c o m m o n m e c h a n i s t i c b a s i s for the r e a c t i o n of the two

enantiomers

(Pig. 2). A c c o r d i n g l y , h y d r o x y l group m i g r a t i o n to C - l is e x p e c t e d to o c c u r not only w i t h the the t r a n s f o r m a t i o n of the

( R ) - s u b s t r a t e but also

in

( S ) - i s o m e r , for w h i c h it cannot

d e t e c t e d e x p e r i m e n t a l l y , and d i f f e r e n t i a t i o n b e t w e e n the and the n e w h y d r o x y l group at C - l takes p l a c e at the

be old

inter-

m e d i a t e stage of the a l d e h y d e h y d r a t e so g e n e r a t e d .

Assuming

a well d e f i n e d s t e r i c course of the r e a c t i o n at the

terminus

of the o x y g e n m i g r a t i o n

(e.g. inversion, as in F i g . 2) and

k e e p i n g in m i n d that d i f f e r e n t H - a t o m s are j u m p i n g to

and

f r o m the c o f a c t o r in the two s e r i e s , it is easy to see that the p o s i t i o n of the m i g r a t e d h y d r o x y l g r o u p in the h y d r a t e

in-

392

t e r m e d i a t e d e p e n d s c r i t i c a l l y o n the C-2 c o n f i g u r a t i o n of the s t a r t i n g m a t e r i a l . S p e c i f i c loss or r e t e n t i o n of t h i s group the c o m m o n p r o d u c t f r o m the two series is best v i s u a l i z e d the o u t c o m e of a s t e r e o c o n t r o l l e d d e h y d r a t i o n of the d i o l m e d i a t e d , say, by a s t r a t e g i c a l l y hydroxyl groups

(Fig.

as

geminal

located basic group

the e n z y m e i n t e r a c t i n g w i t h o n l y one of the two

of

enantiotopic

3).

R e c o g n i t i o n of the d u a l role of the e n z y m e , w h i c h

performs

first as a n i s o m e r a s e and t h e n as a s t e r e o s p e c i f i c h y d r a t e h y d r a t a s e , is i m p o r t a n t

de-

since it a l l o w s to d i s c e r n in its

m o d e of a c t i o n a g e n e r a l feature

(Pig. 4) w h i c h is

t e r i s t i c of m a n y o t h e r c o e n z y m e B^^ d e p e n d e n t (for r e v i e w s

in

see r e f e r e n c e s 11 and

charac-

rearrangements

12).

To a c c o u n t for the a b i l i t y of the e n z y m e to a c c o m m o d a t e

both

e n a n t i o m e r s of p r o p a n e d i o l at a single a c t i v e site a n d to

pro-

cess t h e m t h r o u g h a b s t r a c t i o n of d i f f e r e n t h y d r o g e n a t o m s

a

m o d e l has b e e n p r o p o s e d enantiomeric

(4) in w h i c h p o s i t i o n i n g of the

s u b s t r a t e s is b r o u g h t about t h r o u g h

w i t h three c o m m o n b i n d i n g loci

two

interaction

(Fig. 5). F i x a t i o n of the

m e t h y l g r o u p at one p o i n t and h y d r o g e n b r i d g i n g f r o m the two h y d r o x y l g r o u p s to the r e m a i n i n g f i x e d s t a r r e d p o s i t i o n p a r t s d i f f e r e n t t w i s t s to the

(R)- and

im-

( S ) - s u b s t r a t e . As a

c o n s e q u e n c e , d i f f e r e n t H - a t o m s are e x p o s e d in e a c h case to the r e g i o n o c c u p i e d by the c o e n z y m e . T h e i r a n t i p e r i p l a n a r

rela-

t i o n s h i p to the s e c o n d a r y h y d r o x y l g r o u p m i g h t be e x p e c t e d f a v o u r the s u b s e q u e n t m i g r a t i o n of the

to

latter.

To check o n the v a l i d i t y of t h i s m o d e l a n d e x p a n d our

knowl-

edge of the b e h a v i o u r of s u b s t r a t e s at the a c t i v e site we h a v e f o c u s s e d a t t e n t i o n o n the s e c o n d r e a c t i o n c a t a l y z e d by this e n z y m e , the c o n v e r s i o n of g l y c e r o l into

hydroxypropionaldehyde

(Fig. 6). D e p e n d i n g o n h o w the m o l e c u l e is v i e w e d ,

glycerol

393

COSCoA | H COOH

COOH I H2N-C-H l~\ | H COOH R R R—ipn:-H

Figure 4

Figure 6

^

0H

HH

39 ii

can be considered with equal right a derivative of either

(S)-

or (R)-propanediol. If the compound is indeed able to mimic the behaviour of both enantiomers at the active site, then the enzyme should turn blind with respect to the

enantiotopic

branches of the new substrate,while retaining its ability to select for specific but different H-atoms within each branch. The problem can be investigated with substrate samples doubly labelled with deuterium in a specific branch. These can be made available through reduction of the optically active methyl glycerates

(Fig. 7) and the product of their enzymic

conversion are conveniently analyzed after chemical into the hydroxyacid. Reaction of the sample from

conversion

(S)-gly-

cerate gave an acid in which all of the label was retained in the g-position, whereas the sample from (R)-glycerate led, with a strongly reduced reaction rate in the enzymic step, to an optically active acid monodeuteriated at the a-position. This means that both labelled isomers chose to react exclusively as analogues of (R)- rather than of (S)-propanediol; for one of the isomers the option is taken in spite of an adverse isotope effect! The configuration of the optically active acid from one of the above experiments can be predicted by analogy. Comparison with an authentic specimen of the (+)-acid, prepared as in Fig. 8 by exploitation of a known reaction (13, 14), confirmed that substitution at C-2 is indeed occurring with inversion. Additional support for the analogous behaviour of glycerol and (R-)-propanediol came from two sets of experiments. A sample of glycerol stereospecifically monodeuteriated in its enzymeactive branch was prepared through enzymic reduction of (R)glyceraldehyde

(Fig. 9)- The configuration of this material

was checked by nmr-comparison with the two possible racemic diastereoisomers unambiguosly prepared from a common starting material as outlined. Enzymic conversion of the chiral d,-

395

COOCH,3 I H-C-OH I CH 2 0H

CD2OH I H-C-OH I CHjOH

Li AID/

1)B, 2 -enzrne — » 2) Agr ,0

M

(R)

CH2OH

CH2OH

H-C-OH

>

COOH

H-C-OH

COOCH3

COOH I O-C-H I CHjOH

>

H-C-H

C0 2 0H

CD2OH

(S)

Figure 7

COOH COOH «W^HjN-C-H C CUD O-C-H /\ I HOOC H COOH Y

CHjOH rH2N-t-H

O-C-H I CHjOH

^ ^ Q_I_H I CHjOH W-(S)

Figure 8

OH H—I— D H —OH CH.OH

CHO H—|—OH CHjOH

DC™ C —CH^OR

M,/pa

1,0,1 HCOOH „_)_„ IR-COPtt) H—OH CH,OH DM CHjOR Otó«/B.(CIOJ)J

Figure 9

D—UM CH2OH

396

g l y c e r o l and f u r t h e r c h e m i c a l o x i d a t i o n of the r e s u l t i n g

prod-

uct to the o p t i c a l l y active d-^-acid i n d i c a t e d t h a t all of the label h a d b e e n t r a n s f e r r e d to C-2

(Fig. 10). As in the

case

of ( R ) - p r o p a n e d i o l the e n z y m e is s e l e c t i n g s h a r p l y for the H R - a t o m , u s i n g now the p r o c h i r a l i t y of the a d j a c e n t c e n t e r operating the

for

selection.

In k e e p i n g w i t h the e n z y m i c b e h a v i o u r of (R)-propanediol, the a l d e h y d o o x y g e n a t o m in the p r o d u c t f r o m g l y c e r o l is

expected

to o r i g i n a t e from the m i g r a t i n g s e c o n d a r y h y d r o x y l group

of

the s t a r t i n g m a t e r i a l . To settle this point a sample of g l y c e 18 rol l a b e l l e d w i t h

0 in the c r i t i c a l p o s i t i o n

(Pig. 11) w a s

s u b m i t t e d to the a c t i o n of the e n z y m e and the r e s u l t i n g duct t r a p p e d by r e d u c t i o n w i t h y e a s t - a l c o h o l

pro-

dehydrogenase

18 and N A D H . The

0 - c o n t e n t of the 1 , 3 - d i o l so o b t a i n e d

depends

c r i t i c a l l y on the c o n c e n t r a t i o n of the r e d u c i n g e n z y m e ;

when

t h i s is u s e d in large e x c e s s up to

ob-

r e t e n t i o n can be

no g l u nsample d e r thel a bsame n t0a lin c o n d i te in oz ny s userved. s i n g a Wg o lr yk ci er e l l e de x p we ir ti h m e18 the m e and active h y d r o x y m e t h y l group no r e s i d u a l l a b e l c o u l d be d e t e c t e d in the 18 p r o d u c t . T h e r e f o r e the s u b s t a n t i a l loss of 0 o b s e r v e d in the first e x p e r i m e n t

cannot be due to a lack of d i s c r i m i n a t i o n

the d e h y d r a t i o n step p r o p e r , but r a t h e r r e f l e c t s the culty of t r a p p i n g the o r i g i n a l f o r m of the r e l e a s e d

in

diffialdehyde

in a q u a n t i t a t i v e m a n n e r p r i o r to its r a p i d n o n e n z y m i c

ex-

change w i t h the m e d i u m . The u n p r e d i c t a b l e a b i l i t y of a n e n z y m e w h i c h fails to e n t i a t e b e t w e e n e n a n t i o m e r s and yet u n m i s t a k a b l y

differ-

recognizes

enantiotopic groups when dealing with a symmetrical

substrate

h a d t h w a r t e d so far our a t t e m p t s to s u b s t a n t i a t e the i d e a of a d u a L b i n d i n g m o d e for s u b s t r a t e s at the a c t i v e site. The sults f r o m a d e t a i l e d study of the t h i r d r e a c t i o n

re-

catalyzed

by the e n z y m e , the c o n v e r s i o n of e t h y l e n e g l y c o l into

acetal-

OIH H-C-D H-C-OH CH,OH

n CH,OH

COOH D-C-H CH,OH

Figure 10

-O* OIUtCOffi HHjO_' C|HJOH Eixr» H"c* I *Mt-ADH CmO H-C-tfV» CU, I Iiuwi I I NAOH CH,OCOn> CH,0H CH/JH 100%

V I CHOH I CHjOM

NaBH4

CtufH | CHI CH^JH 38' 44%

CHaO^ CH*OI Eniym« I VsmI-AOH I ^^ CHOH CHj CH» I I N4DH I CHjOH CM,OH CM,0

Figure 11

OH H-C-H CHj-C-H OIH

OI* H Hs-C-H„ H.-C-H, OIH

(R) Figure 12

OIH H-C-H h-c-ch3 OH

39a dehyde, eventually provided the desired breakthrough. Ethylene glycol is another molecule which can be taken as a close relative of either (R)- or (S)-propanediol

(Pig. 12) and

might bind equally well in two possible modes at the active site. If this is the case, the absence of stereochemical

in-

formation at the adjacent center should make it very difficult for our enzyme to differentiate between the enantiotopic H ligands of a given methylene group. The trouble with this substrate resides in the high symmetry of the compound, more specifically in the homotopic relationship of its methylene groups which can be distinguished in their behaviour, if at all, only by imparting to one of them some kinetic bias via isotopic substitution. The feasibility of this approach was tested by following the behaviour of a specimen of ethylene glycol doubly labelled with deuterium in one of the methylene groups (Pig. 13). For better understanding the labelled center will be referred to as the a-position. Such a molecule can be processed by the enzyme in two different ways: migration of deuterium from the a - to the 3-position generates

acetaldehyde

labelled as in B, whereas migration of protium in the opposite direction results in the formation of the species A. If the enzyme is sensitive to the isotope substitution, the first of these two processes will be retarded with respect to the second and this, in turn, will be reflected in an altered ratio of A to B in the product. Trapping the aldehyde as the dimedone derivative and analyzing the intensities of the M + and M + - 15 peaks in the mass spectrum of the latter» a value of 28/72 was detected for the A/B-ratio in the product, corresponding to a value of 2,6 for the apparent isotope effect of the reaction. We could now capitalize on this finding to ascertain what residual stereospecificity was left for the hydrogen abstraction

399

A li r a t i o s c x p e c t c d f r o m :

assumption

(Hl-dj

(Sl-dj

(KS)-dj

H -specilli:

bU 50

U 100

25 75

H ^ - s p c c il'ic

0 100

bO 100

25

33. 3 66. 6

33. 3 66. 6

33. 3 66. 6

25 75

25 75

25 75

46 54

44 56

45 55

nonspec. :

k

"

OO-

nonspee. : 7— - 1 D

found

Figure

75

uoo step. It was c e r t a i n l y g r a t i f y i n g to o b s e r v e a l m o s t

identical

A / B - r a t i o s in the a l d e h y d e s a m p l e s o b t a i n e d f r o m the two e n a n t i o m e r i c forms of the m o n o d e u t e r a t e d g l y c o l , since

this

c o n f i r m e d t h a t the e n z y m e h a d i n d e e d t u r n e d b l i n d a n d w a s no l o n g e r able to select a m o n g e n a n t i o t o p i c H - l i g a n d s .

However,

the value of this r a t i o is r a t h e r e m b a r r a s s i n g , as it fails to d i s c l o s e t h e o p e r a t i o n of the isotope e f f e c t u n c o v e r e d in the p r e v i o u s e x p e r i m e n t . I n fact, none

of the simple

assumptions

s u m m a r i z e d in Fig. 14 and r a n g i n g f r o m a s t e r e o s e l e c t i v e to a c o m p l e t e l y r a n d o m p r o c e s s , be it w i t h a n i n f i n i t e or w i t h no isotope e f f e c t , seems able to a c c o u n t

for t h e s e

r e s u l t s . C o n s i d e r a t i o n of the three h y p o t h e t i c a l

experimental reaction

p r o f i l e s of P i g . 15 p r o v i d e s a w e l c o m e p o s s i b i l i t y f y i n g this u n u s u a l state of a f f a i r s . A s u b s t r a t e

for

clari-

in w h i c h one

of two h o m o t o p i c g r o u p s has b e e n l a b e l l e d w i t h d e u t e r i u m bind with equal probabilities

can

in two d i f f e r e n t w a y s to the

ac-

tive site of the e n z y m e w i t h w h i c h it i n t e r a c t s , thus

offering

at r a n d o m the l a b e l l e d or the u n l a b e l l e d g r o u p to the

subse-

quent a c t i o n of the c a t a l y s t . If the rate of the o n w a r d a c t i o n is m u c h f a s t e r t h a n the d i s s o c i a t i o n of the

re-

ES-complex

(curve I, k j > k ^ ) , t h e n the e n z y m e is f o r c e d to p r o c e s s e v e r g r o u p it was o f f e r e d b e f o r e a c c e p t i n g the next

what-

substrate

m o l e c u l e ; in t h i s case e v e n a l a r g e i s o t o p e e f f e c t w i l l a l t e r the s t a t i s t i c a l d i s t r i b u t i o n of the l a b e l in the

hardly prod-

u c t . A l t e r n a t i v e l y , w h e n d i s s o c i a t i o n of the E S - c o m p l e x

is

f a s t e r t h a n the s u b s e q u e n t r e a c t i o n

the

(curve II, k^ < k ^ )

e n z y m e h a s a chance to r e f u s e the h a r d g r o u p a n d r e a c t

pre-

f e r e n t i a l l y w i t h the light o n e , a n d as a c o n s e q u e n c e the

full

impact of the i s o t o p i c d i s c r i m i n a t i o n w i l l be r e c o r d e d by the label d i s t r i b u t i o n in the p r o d u c t . In t h e i n t e r m e d i a t e

case of

curve III only a f r a c t i o n of the true i s o t o p e e f f e c t w i l l be r e f l e c t e d in the c o m p o s i t i o n of the p r o d u c t . A m o r e v e r s i o n of t h i s scheme for the h a n d l i n g of

detailed

(R)-d..-ethylene-

i»01

Figure 15

,OH

« "

CH,-C (Ai

7 CH,D—C. % ( Bl

O

% (A) calc.

i "f

kHMp= Me .

k

„

k

o

- 10 ;

2 k

H "

»,

- 4

Figure 16

found

d, /f-C-NH(CH;)6NH-{.^ H,0 VOOH) , í n-1 L

amlnohexylamlde-Sepharose 4B

tpa i

VI

Co-Am1noethylcobalam1n-

M

^H 2 CH ; HH-{""j W o /

Co-Carboxyethylcobalamínamlnohexylamlde-Sepharose 4B

VIII

He2Bza

1CH2 I? 0=t(CH 2 ^t-NH(CH 2 ) 6 NH-| Sepharose 4B

VII

structure

CHjCHjü-NH(CHj)6NH-g¡ / c o /

N 6 -Carboxymethyl-Co-adenosylcobalamin-

^

jH^

amlnohexylamlde-Sepharose 4B

NH-CH2C-NHtCH2)6NH^ (L • adenosyl, c y t i d y l , CHj, CN. OH. e t c . )

Fig. 6

Partial structures of Sepharose-bound derivatives of cobalamin

UZG

corrinoid derivatives used in the studies (15). In Compounds I to HI, cobalamin links to Sepharose 4B through the carboxylic acid side chain at position e.

Their upper ligand is adenosyl, cytidyl, methyl, cyano, or hydro-

xyl etc. and the lower ligand is complete or replaced with water.

In the

case of IV, plural propionamide side chains are converted to free acids. In Compound V, cobalamin links to Sepharose at the O p p o s i t i o n of the ribose moiety of the lower nucleotide ligand.

In Compounds VL and V H , cobalamin

binds to Sepharose through an alkyl group attached to Co atom.

Cobalamin

6

is attached at N -position of adenine part of the upper ligand in Compound VHI.

The principle of the affinity chromatography is based on the fact

that the specific binding of AdoCbl and apodiol dehydratase occurs in the presence of K + ion and the absence of the monovalent cation causes the rapid dissociation of AdoCbl due to a drastic conformational change of apoenzyme {loo. ait.).

Hence, the apoprotein bound bio-specifically to immo-

bilized corrinoid derivatives can be eluted with an appropriate eluting

Fig. 7 Affinity chromatography of diol dehydratase on the adenosyl form of Type II Sepharose-bound corrinoid derivatives. About 1 unit of enzyme was applied to the adsorbent (1 ml of packed gel) in 0.1 M K-phosphate buffur (pH 8) containing 2 % 1,2-propanediol, and the affinity chromatography was carried out by using the following eluting agents. Effluent: 0.1 M K-phosphate buffer (pH 8) + 2 % 1,2-propanediol Eluate 1 (El): 0.5 M K-phosphate buffer (pH 8) + 2 % 1,2-propanediol Eluate 2 (E2): 0.3 M Tris-HCl buffer (pH 8) + 2 % 1,2-propanediol Eluate 3 (E3): 0.3 M Tris-HCl buffer (pH 8) Eluate 4: 0.1 M Acetic acid + 2 % 1,2-propanediol

U21

agent not containing K

ion.

Fig. 7 depicts a typical successful elution

pattern by the use of the adenosyl form of Type H

derivatives (AdoCbl

bound to Sepharose through one carboxylic acid side chain (at position e). Apodiol dehydratase adsorbed bio-specifically on the adsorbent is exclusively eluted in the fraction E-2. Analyses of the interaction between apodiol dehydratase and the different types of Sepharose-bound corrinoids provide useful information concerning the structural requirement of AdoCbl molecule.

The results indicate that

some of the side chains of corrin nucleus and the ribose moiety of the lower ligand

will play certain important roles for the binding of AdoCbl

to apodiol dehydratase.

The upper 5'-deoxyadenosyl ligand should take an

indispensable part not only in the binding but also in the activation of the Co-C bond of the coenzyme. 4)

Interaction between apodiol dehydratase and analogs of coenzyme B ^

Accumulated information on the interaction between AdoCbl and apodiol dehydratase suggests strongly that the combined actions of hydrogen bond, van der Waals force, hydrophobic bond etc., at some positions of the corrin nucleus (especially, its B and C rings), some of the aminocarboxyalkyl side chains, and the ribose and base moieties of the lower ligand render the binding of Adocbl to apoprotein very strong and effective in the presence of an effective monovalent cation such as

ion.

Incorporation of halogen at the carbon 10 position of the corrin nucleus had little influence on the affinity for apoprotein, but resulted in a marked loss of the activity of the resulting holoenzyme (16), probably due to an alteration of the electronic structure of the corrin nucleus and further the electronic property of the Co-C bond.

Adenosyl-13-epicobalamin is

an analog in which the adenosyl moiety in the upper coordination position is positioned differently above the corrin ring because the inverted e-propionamide side chain blocks its normal location above C-13 (17).

Further-

more, the lower pK^ of the "base on"^r^"base off" conversion of the analog (2.8) than that of AdoCbl (3.5) suggests that the inversion at C-13 also affects the electronic character of the cobalt atom.

This coenzyme analog

showed approximately 14 % of the coenzyme activity of AdoCbl, while its apparent K^

value (12.7 yM) was 13 times higher than that of AdoCbl for

diol dehydratase (18).

The kinetics of the reaction and the apoenzyme-

adenosyl-13-epicobalamin interaction suggested a lower affinity of the

1+28

No. CH. I

0.

VIII

/ OH

OH -CH^^O. II

CH2

Adenosylcobalamin (Vitamin B , . coenzyme)

Ara-adenosylcobalamin

o o U O

Aristeromycylcobalamin

3-Isoadenosylcobalamin

OH

-CH. Nebularylcobalamin

-CHVI

Inosylcobalamin

Cytidylcobalamin

Adenosylethylcobalamin

3-adeninyl

-CH 2

OH 9-purinyl

• o OH

OH

OH

OH

xy 0H

OH

Adeninylhexylcobalamin Adeninylpenty1cobalamin

-(CH 2 ) 6 -9-aden1nyl

XIII

Adeninylbutylcobalamin

-(CH 2 ) 5 -9-adeninyl

Adeninylpropylcobalamin

- ò

Fig. 8 Partial structures of AdoCbl and its analogs with a modified upper lingand used in this study. The R group represents the nucleoside axial ligand linked covalently to the cobalt atom. affinity of the coenzyme analog for the enzyme.

The analog was readily dis-

sociated even from the reacting apoenzyme-adenosyl-13-epicobalamin complex upon gel filtration.

Like the native holoenzyme, the apoenzyme-coenzyme

analog complex underwent inactivation by oxygen in the absence of substrate. However, the rate of inactivation was much slower than that of the native holoenzyme. With respect to the function of the upper ligand, the 5'-deoxyadenosyl moiety should play the most important role in the activation of the Co-C bond through interaction with the corresponding sites of apoenzyme.

To obtain

more precise knowledge on the function of 5'-deoxyadenosyl ligand, the coenzyme action of analogs shown in Fig. 8 were investigated (19).

1+29

These 14 analogs have a slightly or fairly modified upper ligand.

On the

basis of several lines of criteria concerning the coenzyme activity for diol dehydratase, these analogs were classified into three groups: Type 1, which contains adenine or purine moiety and an intact or slightly modified D-ribose moiety in the upper ligand, such as ara-adenosyl,

aristeromicyl-,

3-isoadenosyl-, and nebularylcobalamins, was able to function as coenzyme, the coenzyme activity decreasing in this order.

Like the native holoenzyme,

complexes of these 4 analogs w i t h apoenzyme showed a cob(IE)alamin-like absorption peak or shoulder in the presence of substrate.

This result means

that: 1) The analog belonging to Type 1 can b i n d effectively to apoprotein to yield an active holoenzyme species; 2) their Co-C bond is activated by the interaction w i t h the apoprotein, and then cleaved homolytically to yield cob(IL )alamin ( B ^

)

anc

^ free radical species of the upper ligand w h e n sub-

strate comes; 3) the enzymic reaction proceeds as in the case of normal coenzyme.

On the other hand, Type 2 analogs, which contain hypoxanthine,

cytosine, and benzimidazole as the nucleoside moiety of the upper ligand in place of adenine, did not function as coenzyme but acted as weak competitive inhibitors against AdoCbl.

Their Co-C b o n d w a s cleaved by photochemical

reaction to give a c o b ( H ) alamin-like spectrum which was unusually towafd oxidation.

resistant

Type 3 analogs, in w h i c h the D-ribosyl moiety is replaced

by L-ribose or by an alkyl chain of 2 to 6 carbons, were inactive as coenzyme and acted as strong competitive inhibitors.

The complexes of these

analogs w i t h apoenzyme showed visible spectra similar to those of free analogs. In conclusion, these results give definitive evidence that b o t h D-ribose moiety and adenine moiety of the upper ligand are essential not only for the effective binding to apoprotein but also for the activation of the Co-C bond of the resulting holoenzyme. Although various interesting features of the interaction between AdoCbl and apodiol dehydratase were delineated from the observations reported in this communication, the mechanism whereby the enzyme activates the Co-C b o n d of AdoCbl still remains to b e elucidated.

Further experimentation on the func-

tional groups at the active sites of the enzyme protein will provide more conclusive information about this interesting phenomenon.

1*30

References 1.

Abeles, R. H., Brownstein, A. M., Randies, C. H.: Biochim. Biophys. Acta, 41, 530-531 (1960).

2.

Lee, H. A. Jr., Abeles, R. H.: J. Biol. Chem. 238, 2367-2373 (1963).

3.

Toraya, T., Shirakashi, T., Kosuga, T., Fukui, S.: Biochem. Biophys. Res. Commun. 69., 475-480 (1976).

4.

Toraya, T., Fukui, S.: Eur. J. Biochem. 21» 285-289 (1977).

5.

Toraya, T., Honda, S., Kuno, S., Fukui, S.: J. Bacteriol. 135. 726729 (1978).

6.

Bradbeer, C.: J. Biol. Chem. 240, 4669-4674 (1965).

7.

Wagner, 0. W., Lee, H. A. Jr., Frey, P. A., Abeles, R. H.: J. Biol. Chem. _241, 1751-1762 (1966).

8.

Stadtman , T. C.: Science, 171, 859-867 (1971).

9.

Toraya, T., Uesaka, M., Fukui, S.: Biochemistry, ¿3, 3895-3899 (1974).

10. Poznanskaja, A. A., Tanizawa, T., Soda, K., Toraya, T., Fukui, S.: Arch. Biochem. Biophys. in press. 11. Schneider, Z., Larsen, E. G., Jacobson, G., Johnson, B. C., Pawelkiewicz, J.: J. Biol. Chem. 245, 3388-3396 (1970). 12. Toraya, T., Sugimoto, Y. , Tamao, Y., Shimizu, S., Fukui, S.: Biochemis t r y , ^ 3475-3484 (1971). 13. Toraya, T., Kondo, M., Isemura, Y., Fukui, S.: Biochemistry, 11, 25992606 (1972). 14. Toraya, T., Ohashi, K., Fukui, S.: Biochemistry, 14_, 4255-4260 (1975). 15. Toraya, T., Fukui, S.: "Methods in Enzymol.", Vol.62B (McCormick, D. B., Wright, L. D., eds.), Academic Press, New York, in press. 16. Tamao, Y., Morikawa, Y., Shimizu, S., Fukui, S.: Biochim. Biophys. Acta, 151, 260-266 (1968). 17. Stoeckli-Evans, H., Edmond, E., Hodgkin, D. C.: J. Chem. Soc., Perkin Trans., U , 605-614 (1972). 18. Toraya, T. , Shirakashi, T., Fukui, S., Hogenkamp, H. P. C.: Biochemistry, 14, 3949-3952 (1975). 19. Toraya, T., Ushio, K., Fukui, S., Hogenkamp, H. P. C.: J. Biol. Chem. 252, 963-970 (1977).

ADENOSYLCOBALAMIN-DEPENDENT GLYCEROL DEHYDRATASE INTERACTION WITH SUBSTRATES AND THEIR ANALOGS

A. A. Poznanskaya and T. L. Korsova A l l - U n i o n Vitamin Research I n s t i t u t e , Moscow, 117246, USSR

Glycerol dehydratase from K l e b s i e l l a pneumoniae ATCC 25955 ( g l y c e r o l h y d r o l y a s e , EC 4 . 2 . 1 . 3 0 ) c a t a l y z e s an i r r e v e r s i b l e conversion of g l y c e r o l , ethylene g l y c o l and 1,2-propanediol

into

propionic aldehydes, r e s p e c t i v e l y ( 1 ) . initial

S - h y d r o x y p r o p i o n i c , a c e t i c and The dependencies of the r e a c t i o n

r a t e s on s u b s t r a t e concentrations f o l l o w the Michaelis-Menten

kinetics.

The

values f o r g l y c e r o l , ethylene g l y c o l and 1,2-propanediol

are ( 1 . 3 - 0 . 0 5 ) - 1 0 " 3 M , ( 0 . 6 0 - 0 . 0 2 ) ' 1 0 ~ 3 M and ( 8 . 0 ^ 0 . 6 ) " 1 0 - 5 M ( T a b l e ) , spectively.

re-

The r a t i o of the dehydration r e a c t i o n rates f o r the above

three compounds at enzyme s a t u r a t i o n with s u b s t r a t e s

constitutes

1:0.4:0.5.

I t has been e a r l i e r shown (2) that f o r 1,2-propanediol

Km i s

virtually

equal to the apparent d i s s o c i a t i o n constant (K Q ) of the enzyme-substrate complex and, t h e r e f o r e , c h a r a c t e r i z e s the true enzyme a f f i n i t y f o r the substrate.

T h i s f a c t g i v e s grounds to reckon that g l y c e r o l a f f i n i t y

g l y c e r o l dehydratase i s almost 15 times l e s s than that of

for

1,2-propanediol,

whereby the ethylene g l y c o l a f f i n i t y i s intermediate as compared to the above two s u b s t r a t e s .

Glycerol dehydratase i s capable of c a t a l y z i n g both ( R ) - and panediol c o n v e r s i o n .

(S)-l,2-pro-

The Km values c a l c u l a t e d from the dehydration

ini-

t i a l rate dependencies on the enantiomer concentrations amount to ( 9 . 6 - 0 . 7 r i 0 ~ 5 M and ( 4 . 0 - 0 . 4 ) - 1 0 " 5 M f o r ( R ) - and ( S ) - l , 2 - p r o p a n e d i o l s , r e s p e c t i v e l y ( T a b l e ) , whereas the conversion rate i s 1 . 9 - f o l d f a s t e r f o r ( R ) - as compared to (S)-enantiomer.

Higher ( S ) - l ,2-propanediol

affinity

f o r g l y c e r o l dehydratase i s apparently i n d i c a t i v e of c l o s e r f i t between

© 1979 Walter de Gruyter &: Co., Berlin • New York Vitamin B l s

£+32 the enzyme active site arid (S)- rather than (R)-enantiomer; however, in respect to catalytic stages, the conformation of (S)-enantiomer: complex seems to be inferior.

enzyme

It is noteworthy that diol dehydratase, the

enzyme sharing with glycerol dehydratase many similar enzymatic properties is also devoid of substrate stereospecificity (3, 4).

Table.

Kinetic parameters for glycerol dehydratase substrates and their analogs

Substrates and analogs 3

K , mM m

V

Glycerol

1.30

1

0.35

Ethylene glycol

0.60

0..4

0.19

(RS)-l,2-propanediol

0.080

0,.5

0.04

(R)-l,2-propanediol

0.096

0..75

(S)-l,2-propanediol

0.04

0..4

1,2-butanediol

, rel.

K n , mM D

k. , min ^ in

2.2

0.02

3-chloro-1,2-propanediol

2.1

0.54

Glycerol methyl ether

4.0

0.05

Glycerol ethyl ether

3.9

0.07

1,3-propanediol

7.0

4.3

1,3-butanediol

150

1.1

1,4-butanediol

110

0.03

With the purpose of studying the glycerol dehydratase substrate specificity, a series of substrate analogs, aliphatic diols differing in the carbon chain length and hydroxyl group disposition were examined.

The

diols under study failed to be glycerol dehydratase substrates, but manifested a competitive inhibition towards the enzyme substrates.

There-

fore, aliphatic diols combine reversibly with the enzyme substrate site forming ternary nonproductive complexes.

k33

The data obtained provide the evidence f o r rather narrow substrate s p e c i f i c i t y of glycerol dehydratase.

The structure of the substrate should

meet at l e a s t two requirements, namely to contain two v i c i n a l

hydroxyls,

and to be composed of a chain not exceeding three carbon atoms.

In addi-

t i o n , a s i g n i f i c a n t role in the c a t a l y s i s belongs to a substituent at the substrate C-3 atom.

The highest rate of glycerol conversion, as compared

to other substrates, implies that the presence

of hydroxyl group at C-3

atom f a c i l i t a t e s the a c q u i s i t i o n of the c a t a l y t i c d l l y most preferable active s i t e conformation.

During the c a t a l y s i s , glycerol dehydratase s u f f e r s an i r r e v e r s i b l e vation (5).

inacti-

E a r l i e r we studied the k i n e t i c s of t h i s process and found

that the i n a c t i v a t i o n observed under conditions of complete enzyme saturat i o n with substrate i s a f i r s t - o r d e r reaction and a r i s e s from the spontaneous i n a c t i v a t i o n of ternary c a t a l y t i c a l l y active enzyme complexes with AdoCbl and substrates (6, 7).

The i n a c t i v a t i o n rate constant ( k ^ )

these complexes i s influenced by the nature of substrates (Table). nonproductive ternary enzyme: a l s o undergo i n a c t i v a t i o n .

for The

AdoCbl complexes with the substrate analogs

From the dependencies of glycerol

dehydratase

i n a c t i v a t i o n rates on the concentration of substrate analogs ( 6 ) , the d i s s o c i a t i o n constants (K^) of ternary complexes and t h e i r

inactivation

rate constants were determined (see Table).

The very f a c t of the ternary complex i n a c t i v a t i o n t e s t i f i e s to t h i s process taking place in the enzyme active s i t e .

Supportive evidence for

t h i s notion was extracted from the studies on the s u b s t r a t e ' s

(1,2-pro-

panediol) e f f e c t s upon the rate of the enzyme i n a c t i v a t i o n in the presence of AdoCbl and 1,3-propanediol.

The experiments were conducted under the

conditions that a l l enzyme taken was bound in ternary complexes, enzyme: AdoCbl: 1,2-propanediol panediol ( k ^ = 4.3 min be neglected.

(k. -1

= 0.4 min" ) and enzyme: AdoCbl: 1,3-pro-

), but i n a c t i v a t i o n of the f i r s t complex could

Then the observed rate constant of glycerol

i n a c t i v a t i o n , k. , .

.« depends on the 1,2-propanediol

dehydratase

( 1 , 2 - P r ) and

1,3-propanediol

(1,3-Pr) concentrations in the following way: k' . in

^in(obsd) [1,3-Pr]

[1,2-Pr]

where K^ and K ^ - d i s s o c i a t i o n constants for 1,2-propanediol and 1,3-propanediol, r e s p e c t i v e l y ; k V n - i n a c t i v a t i o n rate constant for the ternary complex i n v o l v i n g 1,3-propanediol.

The equaiton i s s i m i l a r to those des-

c r i b i n g simple competitive i n h i b i t i o n i f 1,2-propanediol i s regarded as i n h i b i t o r of the i n a c t i v a t i o n .

The k - j ^ p ^ j dependencies on 1,3-pro-

panediol concentration at fixed 1,2-propanediol concentrations are l i n e a r in double reciprocal plots and intersect in one point on the ordinate a x i s (see F i g u r e ) . of the 1,2-propanediol

This indicates the purely competitive nature

i n h i b i t i o n of the i n a c t i v a t i o n of the enzyme:

AdoCbl: 1,3-propanediol complex.

The d i s s o c i a t i o n constant f o r 1,2-pro_5

panediol found from the above dependency i s 7.5*10

M and corresponds to

i t s Km value, thereby implying the occurrence of the c a t a l y s i s and i n a c t i vation at the same s i t e of glycerol

dehydratase.

1/[1,3-propanediol[, Figure.

The 1,2-propanediol effect on the dependence of glycerol dehydratase i n a c t i v a t i o n rate constant on 1,3-propanediol concentration in the presence of AdoCbl. Concentration of 1,2-propanediol: a - 1 mM, b - 2 mM, c - 3 mM, d - 3.5 mM.

Crude g l y c e r o l

dehydratase:OH-Cbl complex was mixed with c e l i t e (50 g per

1 g p r o t e i n ) and then K,,HP04 was added to obtain 1.8 M. The suspension was placed in a g l a s s column and protein f r a c t i o n a t i o n was performed using dec r e a s i n g g r a d i e n t of K„HP0, ( i n 1 pM OH-Cbl) from 1.8 M to 1 M. The com6 8 plex was assayed '

in f r a c t i o n s , pooled and concentrated to 5 - 7 mg pro-

tein/ml u s i n g the vacuum d i a l y s i s technique with S e l e c t r o n u l t r a Thimble ( S c h l e i c h e r & S c h ü l l , Germany). A s o l u t i o n of the complex in 0.5 M I^HPO^ was heated f o r 3 min at 70°C. I t was then cooled in i c e , the pH adjusted to 7 . 5 , and the whole c e n t r i f u g e d . The pH of c l e a r supernatant was r e s e t

© 1979 Walter de Gruyter & Co., Berlin • New York Vitamin B12

i+38

to 8.6 and the complex was f i n a l l y p u r i f i e d ( a f t e r concentration) on B i o gel P-200 column using 0.05 M K 2 HP0 4 in 1 yM OH-Cbl. The r e s u l t i n g complex was free of NAD-dependent glycerol dehydrogenase (EC 1.1.1.6) and stable in 0 . 1 . - 0.5 M K 2 HP0 4 at -20°C. The glycerol dehydratase (sp.act. 40 - 80 ymol/mg-min) can be e a s i l y prepared from the complex by removal of cobalamin. The suspension of 2 - 5 mg charcoal in 0.1 M substrate, 0.05 M K 2 S 0 3 , 0.03 M Mg-acetate, 0.1 M Kphosphate buffer (pH 8.6) and 1 mg of the complex was s t i r r e d f o r 2 - 4

hrs

at 37°C and f i n a l l y centrifuged. The supernatant contained active glycerol dehydratase. The r e a c t i v a t i o n of the complex proceeds with g l y c e r o l , /R/and /S/-1,2-propanediol and ethylene g l y c o l . 8

10~ ) in glycerol reaction: AdoCbl, 6.3 (V ( V r e 1 8 3 ) ; (di-ChloroBza)AdoCba, 5.2 (V

values f o r coenzymes ( i n -jlOO); (NaftBza)AdoCba, 4.0

61)j isoAdoCbl, 6.0

2'-dAdoCbl, 11.0 ( V r e ] 3 4 ) ; 3'-dAdoCbl, 11.0 ( V r e ] 2 5 ) ;

(Vrel48);

(diNitroBza)AdoCba,

3.8 ( V r e 1 2 2 ) ; N g -MeAdoCbl, 25.0 ( V r e l 8 . 5 ) ; N^MeAdoCbl, not a c t i v e .

References. 1. Zagalak, B.: Acta Biochim. Polon., jO, 387 (1963). 2. Zagalak, B. and Pawelkiewicz, J . : Acta Biochim. Polon., VJ_, 49 (1964). 3. Zagalak, B. and Pawelkiewicz, J . : L i f e Sciences, 1962, 395. 4. Pawelkiewicz, J. and Zagalak, B.: Acta Biochim. Polon.,

207 (1965).

5. Zagalak, B.: B u l l . Acad. Polon. S c i . , C I . I I , J 6 , 67 (1968). 6. Zagalak, B.: "Mechanism of coenzyme B ^ action in reactions catalyzed by glycerol dehydratase and diol dehydratase", Monography No.19, Annals of Agr. Univ. in Poznan, H a b i l i t a t i o n Works, Poznan, 1968, 72 pages. 7. A r i g o n i , D., Retey, J . , Bonetti, V., Momtchev, M. and Zagalak, B.: in W. F r i e d r i c h , "Vitamin B ^ und verwandte C o r r i n o i d e " , Georg Thieme Verl a g , S t u t t g a r t 1975, p. 212, 333. 8. Schneider, Z., Larsen, E.G., Jacobsen, G., Johnson, B.C. and Pawelkiewicz, J . : J. B i o l . Chem., 245, 3388 (1970).

THE MECHANISM OF ACTION OF METHYLMALONYL-CoA MUTASE AS STUDIED WITH ISOTOPE LABELLING AND SYNTHETIC MODELS

J. Rätey Lehrstuhl für Biochemie im Institut für Organische Chemie der Universität Karlsruhe, Richard-Willstätter-Allee, 7500 Karlsruhe

Introduction and Outline The isomerisation of methylmalonyl-CoA to succinyl-CoA is the only coenzyme-B^2~dependent rearrangement occuring not only in bacteria but also in higher animals and in man. This process is part of a reaction sequence that enables propionylCoA - a degradation product of branched-chain fatty acids and of certain amino acids, e.g. isoleucine - to enter the citric acid cycle (Scheme 1).

"0

COSCoA

x

epimerase

CH,-C-H

COSCoA

SCoA epimerase

c

II

3"

I

H-C-CHo

d

I

COOH

COOH

CH

(S)

3

n

(R)

c o o h

mutase C02"biotin-enzyme

B]2"CO enzyme COSCoA

COSCoA CH2 I c h 3

^ «*

I isoleucine branched chain fatty acids

Citrate cycle

Scheme 1

© 1979 Walter de Gruyter & Co., Berlin • New York Vitamin B 12

ch2-ch2 COOH

kkO Early studies with appropriately labelled substrates revealed (1, 2, 3) that the isomerisation is brought about by an intramolecular migration of the COSCoA-group to the methyl group. Furthermore, Lynen and colleagues (4) isolated for the first time epimerase-free methylmalonyl-CoA mutase and showed that no isotope incorporation from tritiated water into the product takes place during the mutase reaction. This apparently indicates a hydrogen atom migration from the methyl group to the adjacent carbon atom without exchange with the solvent. More detail about the hydrogen migration became available after discovering the role of the cobalt-bound methylene group of coenzyme-B^2 as a hydrogen carrier (5). /5'- Ej-Coenzyme B ^

was

prepared enzymatically (6) and used as a co-

factor in the methylmalonyl-CoA mutase reaction. With succinyl-CoA as substrate 32% of the tritium, and with methylmalonyl-CoA as substrate 16% of the tritium, was in each case transferred to succinyl-CoA. The fate of the remaining tritium was not investigated.

Another group has reported (7)

24 % tritium transfer in a similar experiment using /5'- Kjcoenzyme B ^ prepared by partial synthesis. The fact that the migrating hydrogen is intermediately transferred to the cobalt-bound methylene group of the coenzyme suggested a transient cleavage of the cobalt-carbon bond that might also be of relevance for the subsequent migration step. Whereas this idea found experimental support by ESR and UV/VIS spectroscopic measurements in the coenzyme-B^2 dependent dioldehydrase (8, 9) and aminoethanol ammonia lyase (10, 11, 12) reactions, no such observations were reported for the methylmalonyl-CoA mutase reaction. Efforts to show a transient paramagnetic vitamin-B^ 2r species were unsuccessful (13, Rétey, J., Konig, A., Wallis, C. , White, H.A., Johnson, A.W., unpublished) but this does not exclude an extremely low stationary concentration of such a species. On the basis of these results the following working hypothesis

i+i+1 for the mechanism of the coenzyme-B^2-catalysed rearrangements was developed:

R'

Scheme 2 An enzyme-induced homolytic cleavage of the cobalt-carbon bond is followed by specific abstraction of the migrating hydrogen atom of the substrate. Rearrangement of the so-formed substrate radical is to all probability catalysed by the central cobalt atom. Although the nature of this catalytic interaction is unknown, transient redox processes between cobalt(II) and the substrate radical have been discussed (14). The rearranged radical is then stabilised by back-transfer of a hydrogen radical from the 5'-methyl group of the 51-deoxyadenosin moiety kept firmly at the active site. Regeneration of the coenzyme by reversal of the very first step terminates the catalytic cycle In order to prove or disprove the above hypothetical mechanism on the one hand and to elucidate further details of the rearrangement on the other, we approached the problem in three different ways; A) by physical and chemical characterisation of the apoenzyme niethylmalonyl-CoA mutase from Propionibac-

kU2 terium shermanii

B) by stereochemical studies using isotopi-

cally labelled substrates and substrate analogues and

C) by

investigating synthetic active site models.

A. Some Properties of the Methylmalonyl-CoA

Mutase

Methylmalonyl-CoA mutase has been isolated on a large scale from sheep liver

(15) and from Propionibacterium

shermanii

(16). The isolation procedures have been described

(15, 16, 17)

and some properties of the two enzymes are compared in Table 1. The mutase from sheep liver has a higher molecular weight than the bacterial enzyme, but both are roughly two orders of magnitude larger than the coenzyme

(mol. weight 1 ,500) . The bacte-

rial enzyme can be dissociated into two different polypeptide chains

(17) and has a lower affinity for coenzyme B ^

than

its conterpart from sheep liver. Table 1. Some physical and chemical properties of methylmalonyl-CoA mutases Source;

sheep liver

(15)

sedimentation constant: molecular weight:

7.7 s

165,000

B ^ - c o e n z y m e bound per molecule: sensitivity to SH-reagents: subunit structure:

2

high

?

Source: Propionibacterium shermanii sedimentation constant: 7.2 molecular weight: 56,000

(18), 7.0 (16), 7.25

(16), 124,000

B 1 2 - c o e n z y m e bound per molecule: sensitivity to SH-reagents: subunit structure:

(16, 17, 18) (17)

(17)

probably 2

minor

after treatment with ammonium

sulphate or guanidine two unequal subunits of molecular weight 60,000 and 65,000 dalton, respectively.

i+t+3

When equilibrium is established the ratio of

(2R)-methylmalo-

nyl-CoA and succinyl-CoA is 1 : 20 (15, 16). One mole of the bacterial mutase catalyses the conversion of 1,400 - 2,000 moles of succinyl-CoA per min. Nothing is known about the primary, secondary or tertiary structures of the mutases. No coenzyme-B.j2 dependent enzyme has yet been crystallised in a form suitable for X-ray studies

(17).

B. Stereochemical and Mechanistic Studies Using

Isotopically

Labelled Substrates and Substrate Analogues All the rearrangements catalysed by c o e n z y m e - B ^

dependent

enzymes involve a substitution at each of the two adjacent carbon atoms constituting the migration termini. The high informative value of the steric course of substitution

reactions

in mechanistic organic chemistry has prompted during the last decade stereochemical studies on various coenzyme-B 1 2

cataly-

sed rearrangements. Whereas retention of configuration has been observed

for the rearrangement of methylmalonyl-CoA

(19, R^tey, J. as cited in ref. 20), the substitution in the corresponding reactions of glutamate 23) and

(S)-/3 -lysine

(21), propanediol

(24) occurs with stereochemical

(22, inver-

sion. In all these cases the steric course was elucidated at only one of the relevant centres and did not allow conclusions to be drawn as to the mechanism of the reaction. The recent finding that ethylmalonyl-CoA is also a substrate for methylmalonyl-CoA mutase from P. sheriranii

(25, 26) paved

the way for more detailed stereochemical studies. When ethylmalonyl-CoA is used as substrate methylsuccinyl-CoA will be formed as product

(Scheme 3). The hydrogen atoms in the sub-

strate and the product which could be involved in the rearrangement are distinguishable and can be specifically belled by deuterium.

la-

kkk COSCoA

COSCoA

I

I

H-C-CH2-CH3

• H 2 c—CH-CH3

COOH

COOH

Scheme 3

COOH

COOR

I

I

CH 2

H-C—COOR

OX

. I 2

1 H — C —2H

H-C-H

I

I

H-C-H

—

I

CH 3

CH 3

CH 3

R--ethyl, (3S)-[3-sH,l ethylraionic acid diethyl ester R=H,(3S)-[3-2Hj ethylmalonic acid

X=H,(1R)-[l-2Hjethanol X=tosyt, (IffJ-fV^Hj ethyl tosylate

-

2

I

H-C-H

I

CH3 X=H, (1S)-|l-2H,]ethanol 2 = tosyl, (1S)-[1' HJ ethyl tosylate

X

,

H-C-COOR

I .

H-C—H

butyric acid

COOH

COOR OX

(3S)-[3- 2 Hj

•

CH 3 R=ethyl,(3R)-[3-JH,] ethylmalonic acid diethyl ester R=H,(3R)-(3-'Hj ethylmalonic acid

I CH2 I ,

-C—H

I

CH 3 (3R)-[3- 2 Hj butyric acid

Scheme 4

The synthesis of stereospecifically labelled ethylmalonic acids (the precursors for the corresponding CoA esters) is outlined in Scheme 4. 2

Enzymatically prepared (R)- and (S)-/1- H^-ethanols (27, 28) were converted to the tosylates and the latter used in a malonic ester synthesis. The expected inversion of configuration was confirmed by decarboxylation of a sample of the 2 derived ethylmalonic acids to the known (3S)- and (3R)-/3- H1/-butyric 2 acids (29). The (3S)- and (3R)-fi- H^-ethylmalonic acids were characterised by ORD and mass spectroscopy and converted to the corresponding CoA esters. These, together with the readily

kk5

2

accessible [2- H^-ethylmalonyl-CoA were rearranged by methylmalonyl-CoA mutase to methylsuccinyl-CoA on a preparative scale. Hydrolysis of the product followed by chromatographic separation of the acid fraction afforded pure samples of methylsuccinic acid which were analysed by ORD/CD, mass and 1 HNMR-spectroscopy (25, 26). The 360-MHz 1H-NMR spectrum of methylsuccinate (Fig. 1) shows a well resolved ABX system corresponding to the methine and the two diastereotopic methylene protons. For the assignment of the methylene protons reference substances of known configuration were needed. The reduction of methyl fumaric and methyl maleic acids by dideuterodiimide occurs in a syn manner 2 (30, 31) and affords threo- and erythro-/"2,3- H_/-methylsucci1 * nic acids, respectively. Examination of the H-NMR spectra of these reference acids led to the assignment of the ABX-quartet at higher field to the proton (HB) in the threo- and of the quartet at lower field to the proton (HA) in the erythro-position with respect to the methyl group (Fig. 1). The analytical tools just described allowed quantitative location of the deuterium in all positions of the enzymically obtained methylsuccinate samples and the results are summarised in Table 2. «B

2!s

'

'

—'

i

f* HT H

TCH5

'

" A

'

'

'

Fig. 1. 360 MHz 1H-NMR spectrum of methylsuccinate

UkG T a b l e 2. C o n v e r s i o n of l a b e l l e d e t h y l m a l o n y l - C o A Products

Substrate H />C0SCoA C'^

„¿•Y

\

CH3 \\ X 2 H ' 7 (S)

COOH

\

L

COOH

2

2 h

7

( R )

\

COOH

3 ^

\OOH -25°/.

IC —

C^HR, V00H

V00H

CH3

-23°/.

COSCoA \

H

H"/

\

H

C H

C^H

COSCoA

-ITU

c>*COSCoA

CH 3

Li

) c —

C 0 0 H

C H

\

c \

H

- 75°/.

COSCoA

^ >C0SCoA

CH3

COSCoA ,C

H " 7 J

H

2H

COSCoA

2

HRe \ ,c

species.

COSCoA

Hsi C^* H Re

CH3

_

\OOH

C O O H

-39'/.

-25°/.

The first a n d s u r p r i s i n g c o n c l u s i o n is the lack of

stereospe-

c i f i c i t y w i t h r e s p e c t to the e n a n t i o m e r i c p u r i t y of the p r o d u c t s . E v e n n o n - l a b e l l e d e t h y l m a l o n y l - C o A as substrate l i s t e d in T a b l e 2) w a s c o n v e r t e d to of only 75% e n a n t i o m e r i c

(not

(2R)-methylsuccinic

acid

purity. 2

E n z y m i c c o n v e r s i o n of ¿2-

H^y-ethylmalonyl-CoA

in d e u t e r i u m

oxide afforded mainly monodeuterated methylsuccinyl-CoA,

the

d e u t e r i u m b e i n g in the 3 - e r y t h r o - p o s i t i o n w i t h r e s p e c t to the 2 - m e t h y l g r o u p . On the a s s u m p t i o n t h a t the m u t a s e w a s s p e c i f i c for

(2R)-ethylmalonyl-CoA

substrate m e t h y l m a l o n y l - C o A )

(as it is for the n a t u r a l

this r e s u l t m e a n s t h a t the

s t i t u t i o n a t C - 2 o c c u r e d w i t h 75% r e t e n t i o n a n d 25% of c o n f i g u r a t i o n . W i t h

stereosub-

inversion

(3S)-f3- H ^ / - e t h y l m a l o n y l - C o A a s i m i l a r

r e s u l t w a s o b t a i n e d , the d e u t e r i u m b e i n g e n t i r e l y r e t a i n e d a t C-3 of the s u b s t r a t e (corresponding to C - 2 of the p r o d u c t ) . 2 When

(3R)-/3- H ^ - e t h y l m a l o n y l - C o A w a s u s e d as s u b s t r a t e

the

o u t c o m e of the e x p e r i m e n t w a s s u r p r i s i n g . O n l y 30 - 40% of the d e u t e r i u m m i g r a t e d to the e x p e c t e d 3 - t h r e o - p o s i t i o n of the prod u c t , in 20 - 25% of the c a s e s it w a s r e t a i n e d at the same C -

it 7 atom to which it had been linked originally and in 35 - 40% of the cases it was substituted by protium. Simultaneously the enantiomeric purity of the product decreased drastically /"(R) : (S) = 60 : 40J. The loss of migrating deuterium was confirmed by the use of [ 2 Hg/- and /"3-2H2/-ethylmalonyl-CoA as substrates. In several experiments 35 - 40% of the migrating deuterium was exchanged to protium during the rearrangement. No deuterium loss was observed in the substrate recovered from the enzymic experiments suggesting that exchange had not occured via ethylmalonyl-CoA formed in an eventual non-stereospecific reverse reaction. In order to inquire into the fate of the lost migrating hydrogen isotope, doubly labelled (3RS)3 14 [3- H,, C(carboxyl)7~ethylmalonyl-CoA was converted to methyl 3 14 succinyl-CoA. Whereas an increase of the H/ C ratio in the recovered substrate revealed a substantial kinetic isotope effect (k. /k3 « 2 - 4) , about 80% of the migrating tritium was " H lacking in the product. This tritium was detected in water. Since on the other hand very little incorporation of tritium into the product could be detected in the complementary experiment carried out in tritiated water (4), the source of the protium that replaces the deuterium lost during migration had 2 2 to be searched for in another way. Therefore [2- H., 3- H,/2

2

methylmalonyl-CoA and [2- H^, 3- ^/-ethylmalonyl-CoA were reacted with the mutase in deuterium oxide. In spite of initial misleading mass spectrometric data, no carbon-bound protium could be detected in the products after examination of their ^H-NMR spectra, thereby excluding the possibility that the CoA-portion of the substrate serves as protium donor. However, upon enzymic rearrangement of /3- H^Z-methylmalonyl-CoA in normal water a significant incorporation of protium into the derived succinic acid was observed (on average one additional proton according to 1H-NMR) whereas much less protium was incorporated into the recovered substrate (70% /^H3/r 16% ¿^H^/, 10% Z^H^J and 4% / ^ H ^ species according to ms, i.e. 0.48 proton on average). These findings are at variance with the pre-

i+i+B viously reported retention of migrating deuterium during the rearrangement (32). Our experiments are, however, the first ones in which both the enzymic reaction and the product analysis were carried out with carrier-free fully deuterated material. These results indicate that in methylmalonyl-CoA holomutase the 5'-CH2-group of coenzyme B 1 2 is not the only site capable of exchanging with the migrating hydrogen atom. A portion of the hydrogen incorporated into the product must originate from the solvent or from a group in the enzymes ' active site which rapidly exchanges with the solvent. The loss of migrating tritium to the solvent may also take place via the same group, however different isotope effects seem to be operative in the "washing in" and "washing out" process.

C. Synthetic Active Site Models Upto a few years ago no chemical analogy existed to the rearrangements catalysed by coenzyme-B^^• Since biochemical evidence suggested direct participation of the coenzyme in the reaction active site , models had to incorporate a cobalt complex. Furthermore, experimental data suggested a transient homolytic cleavage of the Co-C bond of the coenzyme. It became apparent, however, that the free coenzyme cannot catalyse a rearrangement in the same way, because homolytic cleavage of its Co-C bond, induced for instance by irradiation, is an irreversible process. In the presence of oxygen the main product is 5 '-oxo-aaenosine , while under anaerobic conditions a cyclic adenosine derivative is formed in which the 5'-C atom and C-3 of the adenine are directly linked (33). As demonstrated by the following experiment, the photochemical behaviour of free and enzyme—bound c o e n z y m e — c o m p l e t e l y different. Irradiation of a solution of AdoCbl at 0°C led to irreversible cleavage of the Co-C bond within 2 min. Under the same condi-

kk3 tions the complex of AdoCbl and methylmalonyl-CoA mutase was intact - as judged from spectral and catalytic properties even after two hours. This suggested that one of the important functions of the enzyme is to prevent an irreversible cleavage of the Co-C bond by specific binding of both the deoxyadenosine and the cobalamin portion of coenzyme-B12. Such a situation at the active site could be simulated by model cobalt complexes in which the alkyl ligand is held in the ligand sphere of the cobalt either by covalent or non-covalent forces. Our first models, however, consisted of cobalt complexes in which the substrate to be rearranged was simply attached to the central metal atom. Since no biochemical evidence existed for a direct and special role of the corrin ligand in the catalytic process we assumed that it can be replaced by a more simple ligand in a model system. As shown in Scheme 5 methylbrommethylmalonic acid diethylester was reacted with the dime thy lglyoxime complex of cobalt(I), the so-called cobal(I)oxime (34) . The alkylated cobaloxime produced was characterised by spectroscopic methods and elemental analysis and gave upon anaerobic irradiation methylsuccinic acid diethylester in variable yield. The reaction products were identified by glcms. However, the reason for the irreproducibility in the yield of rearranged product could not be elucidated, even after about forty experiments under varying conditions had been performed . When instead of cobaloxime the analogous cobalamin complex was examined, the alkylated derivative was very unstable even in the dark and could not be structurally characterised. However, methylsuccinic acid diethylester has been detected among the reaction products in 1 - 5% yield. Substitution of one of the carboxyethyl groups in the cobalamin complex by a cyano group led to a more stable product that could be purified by chromatography. Exposure of this derivative to day-light for two hours led to its decomposition. Among the products both

i+50

ch3

,

_C02Et -.C0 2 Et

-• C 0 2 E l

CH,

CH 2 Br /CÔT7

Ç02Et

CH 3 ,C02Et Xci>C02Et I çh2 Br

CH 3

3

-C°2Et

\c.>

C0

2

E

.CN CH-5 Xc-^co2E»

«

3

C0 2 Et

I Ç"!

—

I ch 2

CO,Et I 2 I ch 3

2

hv

I CH, |

S> stable

EÉU CH-3

CN .C02Et

C0 2 Et CH^-C-H 1 CH, 1 CN

day

I ch2

light

SM

main / Co /

cobaloxime

kP

cobalamin

product

Scheme oi-methyl- and

/3-methyl

C0 2 Et CH, 1 ch3-c-h CN

CO,Et I 2 CHrC-CN 3 I CH-,

minor product

5

3^-cyanopropionic acid e t h y l e s t e r

t h e f o r m e r in e x c e s s - c o u l d b e d e t e c t e d b y a n a l y t i c a l

-

glc.

T h u s the c y a n o g r o u p h a d a s o m e w h a t h i g h e r b u t c o m p a r a b l e

ten-

dency

and

Kang

to m i g r a t e (35)

than the carboxyethyl

group. Since Scott

(see a l s o P. D o w d in t h i s v o l u m e )

found an

absolute

¡+51

preference of COSR migration against COOR migration the migratory aptitude of these groups appears as follows -COSR> -CN> -COOR. It is generally accepted that anaerobic irradiation of alkylated cobalt complexes leads to an alkyl radical and a paramagnetic cobalt(II) species. In order to test whether the latter plays a role in the rearrangement the radical was generated by a different method. Treatment of methyl-brommethylmalonic acid diethylester with tri-n-butyltin hydride afforded exclusively dimethylmalonic acid diethylester. Similar results were reported by Winstein and coworkers (37) when the homologous radical was generated by decarbonylation of the corresponding aldehyde. All these results suggested a catalytic role for the cobalt atom in the rearrangement. One device that prevents the Fig. 2. The three possible diastereomeric cholestanocpbaloximes.

diffusion of the "substrate" radical away from the ligand sphere of the cobalt is a hydrophobic interaction between the substrate radical and the equatorial ligands. Therefore we introduced the glyoxirae function into ring A of the cholestane skeleton. Cholestane-2,3-dione-dioxime readily formed chiral cobaloximes. As illustrated in Fig. 2 three diastereomeric cholestanocobaloximes can be formed depending on the orientation of the equatorial and axial ligands. Of these, diastereomers A and B have a torsional symmetry whereas diastereomer C is asymmetric. Accordingly, four singlets for the angular methyl groups are observed in the 1H-NMR spectrum of diastereomer C , whereas in the spectrum of diastereomers A and B only two singlets are present (for the spectra of methyl-pyridinocholestanocobaloximes see Fig. 3). Furthermore, in the Fig. 3.1H-NMR spectra of tne diastereomeric cholestanocobaloximes.

J

A_JL

J

lJ

JU

i l ^ ^ M W

i

U53

spectrum of diastereomers A and C, one of the singlets arising from the angular methyl groups are in each case shifted to unusually high field (Fig. 3). These signals must arise from those 19-CH3 groups of the cholestane skeletons that, being close to the axial pyridine ligands experience a ring current effect. In the actual complex forming experiments, all possible diastereomers (A, B and C) were produced, chromatographically separated, and their configurations assigned by ^ H-NMR spectroscopy. One of the axial ligands was invariably pyridine, the other a methylmalonic ester connected to the cobalt by a methylene group (Scheme 6). Irradiation of these complexes in propane-2-ol afforded both dimethylmalonic and methylsuccinic esters. The latter were isolated either by glc (dimethylesters) or hplc and characterised by comparison of their spectra with those of authentic reference compounds. The yields of rearrangement products increased with increasing size and lipophilicity of the alcohol portion of the esters. This is in agreement with the idea that a hydrophobic interaction between the ch

3^C—co2"ch2'r C0 2 -CH 2 -R CHRC-CH3

Cholestane

CC^-CH^R

'""H

CO2-CH2-R +

H-C-CH3 CH 2 CO2-CH2-R yield:

M= Co R=H

Scheme 6

-1 7,

t+54

cholestane skeletons and the axial ligand increases the duration of stay of the photochemically generated radical in the ligand sphere of the cobalt atom thus favouring the rearrangement. In one case the analogous rhodium complex was also prepared and irradiated. The yield of methylsuccinic acid bisnaphthylcarbinyl ester was somewhat lower than in the analogous reaction starting from the corresponding cobalt complex (Scheme 6) . In an effort to hold the generated substrate radical more firmly in the ligand sphere of the cobalt atom the synthesis of cobaloximes was undertaken in which the "substrate" was fixed covalently by tetramethylene bridges to the periphery of the equatorial ligand system. The synthesis of such a complex is described in Scheme 7. Reaction of 5-heptin-1-ol with brommethylmethylmalonyl-dichloride afforded diester that was ozonised at low temperature in methanol and subsequently treated with dimethylsulphide (37). The best yields (up to 80%) of crystalline tetraoxime 2 CH, C H ^ - C s C — ( C H ^ - O H

COCl

CH3

CH2Br

c

c

+

III

III

1

i°3

Exchange of the pyridine ligand against a solvent. Rearrangement?

3

ch

3

ch

3

Z = 0 or NOH

Scheme 7

1*55

Fig. 4. Schematic presentation of the three possible diastereomeric bridged cobaloximes.

were obtained when the resulting crude tetraketone was immediately derivatized (38) . This potentially pentadentate ligand was used as complexing agent for cobalt(II)-chloride in the presence of sodium borohydride and pyridine. In an improved procedure up to 60% yield of a single alkylated cobaloxime 3 was obtained. Fig. 4 shows in a schematic way the three possible diastereomeric monomers to be expected from such a process. The NMR-spectrum of the actual product was compatible only with a cis (B or C) but not with the asymmetric trans configuration (A). Its short term irradiation in methanol led to exchange of the pyridine ligand by a solvent molecule. The resulting complex has been examined by X-ray analysis and its ORTEP diagram is depicted in Fig. 5. The diagram reveals that in the complex-forming reaction the cis-exo configuration (B in Fig. 4) has been realised. Some structural details of the bridged complex are given in Fig. 6. In particular the unusually large (128.5°) Co-C (10>-C (20) angle and the geometry of the angular methyl group are noteworthy. For comparison the analogous angle in coenzyme B 1 2 has been reported to be 125° (39). Irradiation of the bridged model complex for a period of 12 to 24 h under anaerobic conditions in methanolic or ethanolic solution followed by alkaline hydrolysis afforded methylsuccinic acid in 80 - 90% yield (38). This reaction is presently under reinvestigation in our laboratory. Without having

i+56 Fig. 5. ORTEP diagram of the bridged cobaloxime.

Fig. 6. Some structural details of the bridged cobaloxime.

definitive results one can say that the reported results (38) have to be modified and that the reaction is very sensitive to traces of oxygen and to the type of solvent used. In the presence of air the above procedure affords small amounts (up to 10%) of methylmalonic acid. Its formation could be explained by oxygen insertion Fig. 7. Some possible transition into the Co-C bond, state structures. followed by a retro H aldol-type cleavage ,X ,A during the alkaline — cr: hydrolysis. In the ..•¿Col, presence of thioN - y X'N N N phenol dimethylma6 \ lonic acid is the main product. / :

.-Co..,,

W

/ \

N

n -y

xn

In Fig. 7 some of the possible transition state geo-

If57

metries

for the r e a r r a n g e m e n t s

are depicted. A radical

in

w h i c h the m i g r a t i n g g r o u p X i s h a l f w a y b e t w e e n the t w o tion termini

(structure

vourable energetically (AE

40 kcal/mol, r e f .

to be accomodated

migra-

4) h a s b e e n e s t i m a t e d t o b e l e s s than the corresponding c a t i o n i c

fa-

species

4 0 ) , s i n c e the u n p a i r e d e l e c t r o n

in an a n t i - b o n d i n g W a l s h - o r b i t a l .

has

It has

b e e n suggested t h a t p r o t o n a t i o n to form radical c a t i o n 5 could decrease

the transition state energy

sition state structures

(41). T h e l a s t t w o

in F i g . 7 incorporate

cobalt atom. For structure

also the

in w h i c h the three m e m b e r e d

c o n s i s t i n g of t h e m i g r a t i n g g r o u p a n d t h e t w o m i g r a t i o n ni is w i t h its edge a b o v e calculations

the c o b a l t a t o m , e x t e n d e d

have been carried out

ring

termi-

Hiickel

(40) a n d s u g g e s t a

of t h e e n e r g y s t a t e to a p p r o x i m a t e l y the cationic transition

trancentral

lowering

t h e s a m e l e v e l a3 t h a t

state.

W i t h the idea in m i n d that e x p e r i m e n t a l d i f f e r e n t i a t i o n

bet-

w e e n the t w o t r a n s i t i o n s t a t e s 6 a n d ]_ s h o u l d b e p o s s i b l e s y n t h e s i s e d a b r i d g e d a c t i v e s i t e m o d e l i n w h i c h o n e of g r o u p s c a p a b l e of m i g r a t i o n is in the a x i a l p o s i t i o n .

CN 0 H 2 C-(f-0-(CH 2 ) A -C=C-CH 3 —

H0N=C

the in

T // CH3-C=C-tCH^-C-C-0-(CH 2 )^C=C-CH 3

f

*

ra

CH2Br

H0N=C

Scheme

8

(l

we

Complex

8 w a s prepared according to the synthetic p l a n outlined

CN 0 I *

of

í+58 Scheme 8 and spectroscopically characterised. In this complex either the carboxyalkyl group or the cyano group could migrate. In the first case the transition state 6. or in the second its conterpart 1_ should be passed. In the first irradiation experiment we obtained a crystalline product the X-ray structure of which has been determined (Fig. 8). The result reveals that our conditions were far from anaerobic, although the results confirm the assumed structure of our starting material. Apparently in the presence of air oxygen insertion into the Co-C bond is the fastest process. Fig. 8, X-Ray structure of the oxygen insertion product.

Although we cannot yet haviour of our bridged lar active site models coenzyme-B.j 2 catalysed

report a definitive picture of the becomplexes, we hope that these and simiwill help to elucidate the mechanism of rearrangements in more detail.

Acknowledgements: The results from the authors1 laboratory were contributed by Gisela Bidlingmaier, Dr. Helmut Flohr, Dr. Michel Fountoulakis, Dr. Max-Uwe Kempe, Traute Krebs , Alfred Konig, Dr. Edward Smith, Dr. Boleslaw Zagalak and Dr. John A. Robinson who also helped to improve the style of this manuscript. The X-ray work cited was carried out by Drs. Berthold Deppisch and Wolfgang Pannhorst of University

£+59 K a r l s r u h e . W e are g r a t e f u l to D r . W i l l i a m E. Hull of B r u k e r A n a l y t i s c h e M e ß t e c h n i k G m b H , K a r l s r u h e , for r u n n i n g the 360 M H z N M R s p e c t r a and t o the S c h w e i z e r i s c h e N a t i o n a l f o n d s , the Deutsche Forschungsgemeinschaft Industrie for f i n a n c i a l

and the Fonds der

Chemischen

support.

References 1. E g g e r e r , H., O v e r a t h , P . , L y n e n , F., Stadtman, J. Am. C h e m . Soc. 82, 2643-2644 (1960).

E.R.:

2. K e l l e r m e y e r , R.W. , W o o d , H.G. : B i o c h e m i s t r y (1962) .

1124-1131

3. Phares, E . F . , L o n g , M . , C a r s o n , S.F.: B i o c h e m . Res. C o m m u n . 8, 142-146 (1962).

Biophys.

4. O v e r a t h , P . , K e l l e r m a n , G . M . , L y n e n , F . , F r i t z , H . P . , K e l l e r , H . J . : B i o c h e m . Z. 335, 500-518 (1962). 5. F r e y , P . A . , A b e l e s , R . H . : J . B i o l . C h e m . 241, 2732-2733 6. R & t e y , J., A r i g o n i , D. : E x p e r i e n t i a , 22,

783-784

(1966)

(1 966).

7. C a r d i n a l e , G . J . , A b e l e s , R . H . : B i o c h i m . B i o p h y s . A c t a 132, 517-518 (1967). 8. F i n l a y , T . H . , V a l i n s k y , J . , M i l d v a n , A . S . , A b e l e s , R.II.: J. B i o l . C h e m . 248, 1 2 8 5 - 1 2 9 0 (1973). 9. Cockle, S.A., H i l l , H . A . O . , W i l l i a m s , R.J.P., D a v i e s , S.P. F o s t e r , M . A . : J. A m . Chem. Soc. 9±, 275-277 (1972). 10. B a b i o r , B . M . , M o s s , T.H., Gould, D . C . : J. B i o l . C h e m . 4389-4392 (1972).

247,

11. B a b i o r , B . M . , M o s s , T.H. O r m e - J o h n s o n , W . H . , B e i n e r t , H.: J. B i o l . C h e m . 249, 4537-4544 (1974). 12. Joblin, K . N . , J o h n s o n , A . W . , L ä p p e r t , M . F . , Ilollaway, M.R. W h i t e , H.A.: FEBS Lett. 52, 193-198 (1975). 13. Brodie, J.D., W o o d a m s , A . D . , Babior, B.M.: P r o c . 3J_, 1578 (1972) . 14. A b e l e s , R . H . , D o l p h i n , D.: A c c . C h e m . R e s . 9,

Federation 114-120

(1976).

15. C a n n a t a , J . J . B . , F o c e s i , A. Jr., M a z u m d e r , R., W a r n e r , R. C., O c h o a , S.: J. B i o l . C h e m . 2^0, 3249-3257 (1965). 16. K e l l e r m e y e r , R . W . , A l l e n , S.H.G., Stjernholm, R., W o o d , H . G . : J. B i o l . Chem. 23£, 2 5 6 2 - 2 5 6 9 (1964). 17. Zagalak, B . , R e t e y , J., Sund, H.: Eur. J. B i o c h e n . 529-535 (1974).

44,

18. O v e r a t h , P . , Stadtman, E.R., K e l l e r m a n , G.M., L y n e n , F . :

it60 Biochem. Z. 336, 77-98 (1962). 19. Sprecher, M., Clark, M.S., Sprinson, D.B.: J. Biol. Chem. 241 , 872-877 (1966) . 20. Arigoni, D., Eliel, E.L.: Top.Stereochem. 127-243 (1969. 21. Sprecher, M., Switzer, R.L., Sprinson, D.B.: J. Biol. Chem. 2_41_, 864-867 (1966). 22. Retey, J., Umani-Ronchi, A., Seibl, J., Arigoni, D.: Experientia 22, 502-503 (1966). 23. Zagalak, B., Frey, P.A., Karabatsos, G.L., Abeles, R.H.: J. Biol. Chem. 241_, 3028-3035 (1966) . 24. Retey, J., Kunz, F., Arigoni, D., Stadtman. T.C.: Helv. Chim. Acta 61_, 2989-2998 (1 978). 25. R^tey, J., Zagalak, B.: Angew. Chem. £5, 721-722; Angew. Chem. Int. Ed. V2 , 671-672 (1973) . 26. Retey, J., Smith, E.H., Zagalak, B.: Eur. J. Biochem. 83, 437-451 (1978). 27. Günther, H., Biller, F., Kellner, M., Simon, H.: Angew. Chem. 85 , 1 41-142; Angew. Chem. Int .Ed. 1_2 , 1 46-147 (1973). 28. Günther, H., Alizade, M.A., Kellner, M., Biller, F., Simon, H. : Z. Naturforschung 28_, 241-246 (1973). 29. Bücklers, L., Uirani-Ronchi, A., Retey, J., Arigoni, D.: Experientia 26, 931-933 (1970). 30. Corey, E.J., Mock, W.L., Pasto, D.J.: J. Am. Chem. Soc. 83, 2957-2958 (1961 ) . 31. Hünig, S., Müller, H.R., Thier, W. : Tetrahedron Lett. 353-357 (1961). 32. Erfle, J.D., Clark, J.M. Jr., Nystrom, R.F., Johnson, B. C., J. Biol. Chem. 239., 1920-1924 (1964). 33. Hogenkamp, H.P.C.: J. Biol. Chem. 238, 477-480 (1963). 34. Schrauzer, G.N., Kohnle, J.: Chem.Ber.97, 3056-3064 (1964). 35. Scott, A.I., Kang, K.: J.Am.Chem.Soc.9£, 1997-1999 (1977). 36. Lewis, S.N., Miller, J.J., Winstein, S.: J. Org. Chem. 37, 1478-1485 (1972). 37. Re, L., Maurer, B., Ohloff, G.: Helv. Chim. Acta 56_, 18821894 (1973) . 38. Flohr, H., Pannhorst, W., Retey, J.: Helv. Chim. Acta 61, 1565-1587 (1978). 39. Lenhert, P.G.: Proc. Roy. Soc. A303, 45-84 (1968). 40. Salem, L., Eisenstein, 0., Anh, N.T., Bürgi, H.B. Devaguet A., Segal, G., Veillard, A.: Nouveau J. Chim. 1, 335-348 ' (1977). 41. Golding,B.T., Radom,L.: J.Am.Chem.Soc.98, 6331-6338 (1976).

RECENT STUDIES ON THE MECHANISM OF ACTION OF ETHANOLAMINE AMM3NIA-LYASE.

Bernard M. Babior Frcm Blood Research Laboratory, Tufts New England Medical Center Hospital Boston, MA 02111

Ethanolamine anroonia-lyase is an AdoCbl-requiring enzyme which catalyzes the conversion of ethanolamine and propanolamine to amnonia and the respective aldehydes (1,2). In the course of the reaction, the amino group exchanges places with a hydrogen on the carbinol carbon of the substrate, producing a 1-amino alcohol (2,3); this eliminates anmonia in a subsequent step to yield the final products (Equation 1) . The ethanolamine anmonialyase reaction is thus similar to other AdoCbl-dependent rearrangements, all of which occur by the migration of a hydrogen atcm frcm one carbon to an adjacent one in exchange for a group X, which migrates in the opposite direction (4,5).

H2N

OH

CH2-CH — • H

NH 2 HCH2-CH

NHG ^

•

HCH2CHO

OH

Equation 1 Although these rearrangements have been studied intensively in an attanpt to shed light on their mechanisms, it is probably fair to say that they are less well understood than almost any other group of enzyme-catalyzed reactions. Nonetheless, certain insights have been obtained through experiments carried out in several laboratories over the past 15 years (4, 5). These largely deal with the mechanism of hydrogen migration. According to current thinking, hydrogen transfer involves AdoCbl as an intermediate hydrogen carrier, and takes place as shown in Equation 2. In this scheme, hemolysis of the C-Co bond is followed by the transfer of a hydrogen atom frcm the substrate to the deoxyadenosyl fragment to produce __

© 1979 Walter de Gruyter & Co., Berlin • New York Vitamin B12

U62

51-deoxyadenosine and a substrate radical. This rearranges to a product radical, which is then converted to the final product by the reverse of the above steps. While the foregoing schene provides a satisfying, if elementary, explanation for hydrogen transfer, it leaves unanswered many questions concerning these rearrangements. Three of these questions will be dealt with in this report. The questions to be addressed are: a) what drives the hemolysis of the C-Co bond? b) how does the substrate radical rearrange? and c) what does stereochanistry say about the reaction mechanism? What drives the hemolysis of the C-Co bond? Hemolysis of the C-Co bond of AdoCbl is clearly the effect of a force exerted on the cofactor by the

RCH2

OH

RCHJH

I

+

H-C-CH2NH2

H

zc¡7

OH

I

+ -C-CHJNHJ

H

/Co7

RCH,

/ci7 RCH2

OH

I

/Co7

+

HCH2-CH NH 2

RCHJH

OH +

/Co7

I I

'CHJ-CH NH 2

Equation 2 enzyme, since AdoCbl alone is stable in solution at rcon temperature as long as it is shielded frcm light. In other systans, it has been shown that an enzyme operates in part by applying distorting forces to the substrate which carpel it to assume a strained conformation resembling that of the transition state of the catalyzed reaction (6). Experiments with a series of AdoCbl analogs suggested that such distorting forces—specifically, forces which alter the conformation of the corrin ring—are involved in the catalysis of C-Co bond hotiolysis. The analogs employed in these experiments were a series of adeninylalkyl

463

oobalamins in which an adenine is attached to the cobalt by methylene bridges whose lengths varied frcm 2 to 6 carbons (Diagram). All these analogs were shown to bind to the active site of the enzyme, since all were found to be inhibitors competing kinetically with AdoCbl. However, pronounced differences were observed among the analogs in the way in which they interacted with the enzyme. The conclusion that alterations in the conformation of the corrin ring drive the hemolysis of the C-Co bond is based on these differences. In one series of experiments, the effect of illumination of the enzymeanalog complexes was investigated. The results are shown in Table 1.

NH 2

ZH7 Table 1.

Adeninylalkylcobalamin

Effect of light on enzyme-bound adeninylalkylcobalartuns. Cobalamin

n

Product

(w-AdeEt) Cbl (oj-AdePr) Cbl (w-AdeBu)Cbl

2 3 4

Cbl(II) Cbl (II) No change

(w-AdePe)Cbl

5

(w-AdeHx)Cbl AdoCbl

6 —

Cbl(II)

The short-chain analogs (n = 2,3) were found to cleave homolytically to enzyme-bound cob(II)alamin, while the long-chain analogs were stable to light. AdoCbl behaved like a short-chain analog. These findings suggest that net hemolysis of the enzyme-bound analog requires light-induoed C-Co cleavage followed by a conformational adjustment which prevents recombination of the fragments by removing the alkyl radical from the vicinity of

k6U the metal. For same reason, this adjustment occurs with the short-chain analogs, and with MoCbl, but not with the long-chain analogs. The nature of the adjustment was suggested by circular dichroism spectroscopy of the enzyme-analog complexes (Figure 1). Clearcut differences were induced in the CD spectra of the long-chain analogs on binding to the enzyme, while the spectra of the C-2 analog and AdoCbl shewed little alteration (the C-3 analog showed intermediate behavior). This suggests that the binding of the long-chain analogs to the enzyme is associated with a change in the ring conformation which does not occur on binding of either the C-2 analog or AdoCbl. These observations can be explained by postulating that the enzyme attempts to force a conformational change on any oorrin ring that binds to the active site. With the long-chain analogs, the preferred conformation can be achieved. With short-chain analogs or with AdoCbl, however, crowding of the Co-S-substituent into the ring prevents this conformational change frcm occurring. The CD spectra of the latter therefore change little on binding to the enzyme, while those of the long-chain analogs show distinct alterations. From these findings, it is reasonable to spec-

/\

1' /

400

J

"

if

El

900

*

f\ /

\*

V' ' " " 'V • 400 WAVELENGTH, m

800

Figure 1 Circular dichroism spectra of free and enzyme-bound cobalamins. The spectra of enzyme-bound (u>-Ade Bu)- and (w-Ade Hx)Cbl were indistinguishable from that of (to-Me Pe)Cbl.

k&5 ulate that enzyme-bound long-chain analogs are stable to light because, the preferred conformation having been achieved, there is no force to prevent the light-cleaved fragments frcm recombining. With the other cobamides, however, the relief of crowding on C-Co bond cleavage permits the conformational change to occur, separating the hcmolyzed fragments so that net photolysis is seen. I therefore propose that C-Go bond hemolysis is forced in part by an enzyrte-driven change in the conformation of the corrin ring which can only occuf when the bulky Co -g-adenosyl residue is separated frcm the metal. How does the substrate radical rearrange? The most puzzling aspect of MoCbl-dependent rearrangements is the mechanism by which group X (~NH2, in the case of ethanolamine airmonia-lyase) migrates frcm one carbon to the next. There is very little biochatiical information concerning this point. This informational void has been filled by a large and growing population of model reactions which now exceed in number even those proposed to explain the mysterious pyrrole rearrangement during porphyrin biosynthesis (7). A feature of many of these models is the intermediacy of a cobalamin derivative produced by the caribination of the substrate radical with the cob(II)alamin at the active site (henceforward, designated "substratocobalamin"). Indeed, I myself postulated such an intermediate to explain the conversion of ethanolamine to 1-aminoethanol (8) (Equation 3). Several recent lines of evidence, however, have indicated strongly that, at least with the ethanolamine ammonia-lyase reaction, such an intermediate cannot participate in group X migration. NH3 HO

I

HC-CH2NH2

Zc°7

HO •

I

HCYCH2

Zc°7 Equation 3

NH2 HC-CH2

I I HO/CT?

U66

Hie first such line of evidence was the result of an experiment designed to test directly whether substratocobalamin (actually "productocobalamin", since the ccrrpaund tested was the hypothetical rearrangement product) was a participant in the enzyme-catalyzed reaction (9). In this experiment, the constituents of the postulated intermediate species (enzyme, 5'-deoxyadenosine and productocobalamin (B-anúiio-6-hydroxyethylcobalamin)) were incubated together with the idea that they might assemble and then undergo the final steps of the catalytic reaction to release acetaldehyde and ammonia and regenerate the coenzyme, which could be assayed directly (Equation 4). Analysis showed, however, that less than 0.0006% of the added cobalamin was converted to catalytically active AdoCbl, a finding which, while constituting negative evidence only, is nevertheless consistent with the notion that substratocobalamin is not an intermediate in the ethanolamine ammonia-lyase reaction. N H2,

I

HC-CH, I | HO / C o 7

Ft

+

NHz

I

CH,

I

Enzyme

HC-CH3 I no

r

+

I

CH 2 J Zca7

»

Equation 4 The second line of evidence was obtained by studying the reaction between enzyme, AdoCbl and aminoacetaldehyde (10). The latter is a substrate analog which promotes enzyme-dependent cleavage of the C-Co bond of the cofactor. It seemed likely that if substratocobalamin were an intermediate in the reaction with the true substrate, then glycylcobalamin, an isolable compound of known properties, should have been formed from aminoacetaldehyde by the path shown below (Equation 5). What was actually observed was the destruction of the cofactor and the conversion of aminoacetaldehyde to acetic acid. There was no indication whatsoever that glycylcobalamin was ever formed. In this reaction, then, the amino group departed from the methylene carbon without the participation of substrat(analog)ocobalamin. This finding represents further evidence against substratocobalamin as an intermediate in the catalytic mechanism. The strongest evidence against substratocobalamin, however, was obtained through studies with 1-aminoisopropanol

. This compound proved to

be a true substrate, though very poor, for ethanolamine aitinonia-lyase, re-

¿+67 acting to yield acetone and (probably) armonia. If as seems likely the reaction path for this substrate is similar to that for ethanolamine, one of the first intermediates would be a substrate radical which is almost isosteric with the t-butyl radical: OH ch3-C-CH2NH2 Such a radical vrould be far too hindered to react with cob(II)alamin to form substratocobalamin; nevertheless, it is cleanly deaminated by the eno" i R H2NCHJ-C-H + *CH2 . ¿5a7

o " +r CH, i • HjNCHJ-C. /"co~7

iio • H2NCHJ_C I /co7

Equation 5 zyme-oofactor complex. Analogous results have been obtained with diol dehydrase (11,12). On the basis of the foregoing evidence, I believe that, whatever the mechanism of amino group migration in the ethanolamine airmonia-lyase reaction, it does not involve substratocobalamin. What does stereochemistry say about the reaction mechanism? Stereochsnical considerations have been used by many workers to draw conclusions concerning the mechanisms of a variety of enzyme-catalyzed reactions (13). For example, a large number of aliphatic hydroxylases catalyze reactions in which -H is replaced by -OH with retention of configuration. These reactions have been postulated to involve the insertion of an oxenoid oxygen atcm into a C-H bond, in part because their stereochemical course is the same as that observed in the C-H insertion reactions of carbenes and nitrenes. The stereochemistry of hydrogen transfer in aldose-ketose isomerizations has been used as evidence to support the idea that a single electrophile at the active site is able to polarize the carbonyl group of either enzyme-bound sugar. Recently, mechanistic studies of phosphate-transferring enzymes have been carried out based on the stereochemical course of reactions in which chirally labelled phosphate, either by itself or incorporated into a nucleotide, has been used as substrate (14).

i+GB lb draw mechanistic inferences from stereochemical considerations for a class of enzymes, however, it is generally necessary that the observed stereochemistry be the same for all enzymes of the class. By this criterion, enzymes catalyzing AdoCbl-dependent rearrangements are completely unsuited for this type of analysis. As a group, these enzymes are noted for their stereochemical unpredictability. Glutamate mutase, for instance, catalyzes an exchange of places between a glycine residue and a hydrogen atom with inversion of configuration at each of the carbon atoms which receive the migrating groups (15), while methylmalonyl CoA mutase catalyzes a similar reaction with retention of configuration at both carbon atons (16). Diol dehydrase ressnbles glutamate mutase in that the configuration of the carbon receiving the migrating hydrogen atern during the dehydration of propylene glycol is inverted. This enzyme, however, possesses the very unusual feature that it is able to use both (R) - and (S)- propylene glycol as substrate (17). Frcm the foregoing, it is clear that general conclusions regarding the mechanism of AdoCbl-dependent rearrangements cannot be drawn from stereochemical considerations. Hcwever, aspects of the mechanism of action of specific enzymes can be elucidated in this fashion. For this reason, an investigation of the stereochemistry of the ethanolamine amraonia-lyase reaction has been undertaken. Seme time ago it was shown that the abstraction of hydrogen frcm the substrate was stereospecific (3)—not a surprising result—although which of the two enantiomeric hydrogen atoms participates in this reaction has not yet been determined. Much more interesting was the result of a study to determine the stereochemical fate of the carbon to which the migrating hydrogen is transferred. This carbon, which becomes the methyl carbon of acetaldehyde, is racemized during the reaction (18), a very unusual outcome for enzyme-catalyzed displacement reactions. The racemization observed in this study was interpreted as evidence for the participation of the symmetrical 2-oxoethyl radical as a catalytic intermediate, in O hc-ch2" 2-oxoethyl radical

£•69 agreanent with a large body of evidence implicating species with unpaired electrons as intermediates in AdoCbl-dependent rearrangements. Mare recent studies have been concerned with the stereochemistry of the ethanolamine aitmonia-lyase catalyzed deamination of 2-aminopropanol. It has been known for sane time that (S)-2-amiiio-propanol is a substrate for ethanolamine amtonia-lyase, rearranging to form propionaldehyde and armonia at a rate approximately 1% that observed with ethanolamine (19). Recent studies have disclosed that (R)-propanolamine is also a substrate, reacting about one-sixth as rapidly as (S)-propanolamine (Table 2). Ethanolamine arrmonia-lyase is thus similar to diol dehydrase in that it can accept either enanticmer of 2-aminopropanol as substrate, although it is more selective than diol dehydrase, which dehydrates (R)- and (S)-propylene glycol at nearly equal rates. Table 2. Rates of deamination of the enanticmers of 2-aminopropanol. Enantianer

Turnover number (sec )

(S)-2-aminopropanol

1.0

10.7

(R)-2-aminopropanol

0.17

2.2

The mechanism of deamination of thetoroenanticmers, however, may differ, at least insofar as the rate-limiting step is concerned. This conclusion is based cm the magnitudes of the deuterium isotope effects for thetarosubstrates, as determined by comparing the rates of deamination of (R) — and (S)-[l,l- D ^ propanolamine with those of the corresponding unlabelled canpounds. The deuterium isotope effect for the (S)-enanticmer was clearly a primary isotope effect (Table 2), indicating that hydrogen transfer was rate-limiting with this substrate as it is with ethanolamine (20). Hcwever, a much smaller isotope effect was seen in the case of the (R)-enantianer. This isotope effect is so small that a step not involving hydrogen transfer would seem to be at least partially rate limiting. The identification of this step by the characterization of the enzyme-bound intermediates which accumulate during the deamination of (R)-2-aminopropanol could provide an important clue regarding the mechanism by which these perplexing rearrangements are accorpi ished.

1+7G

References 1. Kaplan, B.H. and Stadtman, E.R.: J.Biol.Chem. 243, 1789-1793 (1968). 2. Carty, T.J., Babior, B.M. and Abeles, R.H.: J.Biol.Chem. 249, 16831689 (1974). 3: Babior, B.M.: J.Biol.Chem. 244, 449-456 (1969). 4. Babior, B.M.: Account.Chem.Res. 8, 376-384 (1975). 5. Abeles, R.H. and Dolphin, D.H.: Account.Chem.Res. 9, 114-120 (1976). 6. Jencks, W.P.: Adv.Enzymol. 43, 219-410 (1975). 7. Battersby, A.R. and McDonald, E.: Account.Chem.Res. j2, 14-22 (1979). 8. Babior, B.M.: J.Biol.Chem. 245, 6125-6133 (1970). 9. Krouwer, J.S. and Babior, B.M.: J.Biol.Chem. 252, 5004-5009 (1977). 10. Krouwer, J.S., Schultz, R.M. and Babior, B.M.: J.Biol.Chem. 253, 10411047 (1978). 11. Toraya, T., Shirakashi, T., Kosuga, T. and Fukui, S.: Biochem.Biophys. Res.Comm. 69, 475-480 (1976). 12. Bachovchin, W.W., Eagar, R.G., Moore, K.W. and Richards, J.H.: Biochemistry J6, 1082-1092 (1977). 13. Hanson, K.R. and Rose, I.A.: Account.Chem.Res. 8, 1-10 (1975). 14. Eckstein, F. and Goody, C.S.: Biochemistry J_5, 1685-1691

(1976).

15. Sprecher, M. Switzer, R.L. and Sprinson, D.B.: J.Biol.Chem. 241_, 864867 (1966). 16. Retey, J. and Zagalak, B.: Angew.Chem.Int.Ed. YZ, 671-672 (1973). 17. Zagalak, B., Frey, P.A., Karabatsos, G.L. and Abeles, R.H.: J. Biol. Chem. 241_, 3028-3035 (1966). 18. Retey, J., Suckling, D.J., Arigoni, D. and Babior, B.M.: J.Biol.Chem. 246, 6359-6360 (1974). 19. Carty, T.J., Babior, B.M. and Abeles, R.H.: J.Biol.Chem. 249, 16831688 (1974). 20. Weisblat, D.A. and Babior, B.M.: J.Biol.Chem. 246, 6064-6071

(1971).

STUDIES ON THE MECHANISM OF REACTIONS CATALYSED BY ETHANOLAMINE AMMONIALYASE

M. R. Hollaway, H. A. White Department of Biochemistry, University College, London, UK K. N. Joblin, A. W. Johnson, M. F. Lappert and 0. C. Wallis School of Molecular Sciences, University of Sussex, Falmer, Brighton, UK

The recognition of the function of the vitamin B ^ coenzyme, adenosylcobalamin or AdoCbl as the co-factor of a group of at least ten enzymes capable of effecting the rearrangement of substrates which may be electronically unactivated, has evoked much study (reviews see 1 , 2 ) . earlier work on the structure, synthesis and reactions of the B ^

Our coenzyme

(review see 3) had provided us with a background for work on the enzyme systems themselves, but it was clear from the outset that any study of the rapid enzymic reactions was going to depend heavily on physical

techniques.

We chose ethanolamine ammonia-lyase (EAL) as the B ^ enzyme for our studies on account of the relative ease of its preparation and the fact that a single protein is involved in the catalysis.

EAL, first described by

Kaplan and Stadtman (4) is associated particularly with Professor Babior (5) and we wish to acknowledge the numerous

discussions and help we have

had from him on several occasions.

The enzyme, EAL, for these studies was prepared by a modification of the method of Kaplan and Stadtman (4).

Differences between enzyme prepara-

tions from Bethesda, Maryland and Sussex are associated with the quality and quantity of the coenzyme rather than the protein.

The enzyme had been

claimed to have M, 520,000 (4), to contain 2 active sites/mol. (6) and to be composed of 8-10 sub-units, each of M, 51,000 (4).

We have re-examined

the enzyme sub-unit structure using sodium dodecyl sulphate-acrylamide gel electrophoresis and find there are 2 sub-units, M, 51,000 and 36,000 respectively in equimolecular proportions (7).

Each molecule of the native

enzyme is made up of 12 sub-units, 6 of each type.

© 1979 Walter de Gruyter & Co., Berlin • New York Vitamin Bis

Amino-acid analyses

U72

using Edman degradations have indicated an N-terminal sequence Met-IleuLeu for the larger sub-unit and Met-Phe-Ser for the smaller sub-unit. Kinetic analysis, based on results using rapid-scanning, stopped-flow spectrophotometry (see later) of the EAL-catalysed rearrangement of L-2aminopropanol under conditions of high AdoCbl concentration so that full saturation of the enzyme with coenzyme was achieved, has led us to the conclusion that there are 6 functional active sites per molecule of EAL (8).

This correlates more easily with our picture of the sub-unit

structure (I)g-(II)g than the earlier findings (6) of only 2 active sites/ mol. which was based on titration studies and kinetic measurements using 2-aminoethanol as substrate.

Ethanolamine ammonia-lyase catalyses the formation of acetaldehyde and ammonia from 2-aminoethanol or of propionaldehyde and ammonia from L-2aminopropanol in reactions which involve migration of an amino group (which may be protonated) between substrate carbon atoms.

The views of

several research groups, including ourselves, on the mechanism of the rearrangement of 2-aminoethanol and related substrates is summarised in Scheme 1 where substrate-metal binding during the rearrangement may or may not be involved.

The homolytic fission of the cobalt-carbon bond in the enzyme caused by the approach of the substrate (Scheme 1) can also be achieved in the

B^

enzymes or coenzyme, as well as in a variety of other compounds containing cobalt-carbon bonds, by irradiation.

Although the light-induced

homolysis of the coenzyme had been postulated many years ago (9), we have demonstrated this unequivocally by spin trapping experiments (10) using nitroso compounds, RN0(R=Bu t or 2,3,5,6-Me^CgH).

The nitroxides,

Bu N(0)R' so formed from AdoCbl (e.g. I) or EtCbl and Bu t N0 in water at t

50° in the cavity of an e.s.r. spectrometer were characterised by their spectra (see Figure 1).

The B 1 ?

moiety was characterised by a broad e.s.r. signal at g ca. 2.2

i.73

HO ¿HO- CHO R-CH2

R-CH 2

NH3 CHO-CH 3 * OH R-CH2

NH I 3 ¿H2-CH2

H O

R-CH2

CH 3 -CHO+NH 3 ^ ^ + NH3 R-CH 3 CH 2 -CH

HO

NH 3

|NH 3 R-CH 3

NH 3 HO-CH 2 CH 2 -CH R-CH3 CH IOH C o (in)

\

Co('»)

Scheme 1 Mechanistic scheme for the rearrangement of 2-aminoethanol catalysed by EAL. RCH2=5'-deoxy-5'-adenosylcobalamin. The catalytic steps of the reaction are believed to proceed by a mechanism which may (stippled arrows) or may not involve substrate-metal binding during rearrangement. A similar scheme can, in principle, be drawn for L-2-aminopropanol as substrate. upon freezing the irradiated sample to 173K and the nitroxides (e.g. I) were unambiguously identified by the form and line-width variations in the 1 2 e.s.r. spectrum. a-Methylene protons (H and H ) of a nitroxide with a

klk

e - o p t i c a l l y active c h i r a l centre RN(0)CH 2 *CXYZ(X t Y f Z) are magnetically non-equivalent and thus give r i s e to a 1:1:1:1 quartet with a s e l e c t i v e broadening of the inner pair , rather than the 1:2:1 t r i p l e t observed when the B-carbon i s not c h i r a l .

Furthermore the spectrum (Figure 1) shows a

small s p l i t t i n g a t t r i b u t a b l e to hyperfine o (H ) on the 3-carbon atom.

coupling with the hydrogen

Figure 1. E . s . r . spectrum of BuSl(0)R ( R = 5 ' - d e o x y - 5 ' - a d e n o s y l ) at 50°. Peaks marked * are due to Bu^NO.

in H~0

The Ado radical i s thus an intermediate both in the enzyme-induced rearrangements and the anaerobic photolytic transformations of the enzyme, although for the l a t t e r process there i s evidence at low temperatures f o r e . p . r . undetectable i n c i p i e n t homolysis' of the cobalt-carbon bond (11). Thus, p h o t o l y s i s of frozen (80-200K) anaerobic s o l u t i o n s of AdoCbl in aqueous propan-1,2-diol mixtures produced only small Co(11) e . p . r .

sig-

nals but on warming these s o l u t i o n s without further photolysis the Co(11) signal increased 6 - f o l d .

Several workers have examined the p o s s i b i l t y of

e f f e c t i n g rearrangement of the B 1 ? enzyme substrates by mixing them with

475

coenzyme or other compounds containing cobalt-carbon bonds followed by irradiation.

We found (12) that, contrary to an earlier claim (13),

AdoCbl is essentially inert under conditions of anaerobic irradiation at 30°C towards either 2-aminoethanol or ethylene glycol because of the more facile intramolecular cyclisation to 8,5'-cyclo-5'-deoxyadenosine, identified by direct comparison with a sample prepared (9) by photolysis of substrate-free AdoCbl.

On the other hand, similar photolysis of 8-methoxy-

5'-deoxy-5'-adenosylcobalamin

(II), MeCbl, or methyl(aquo)cobaloxime

causes the transformation of the above substrates into acetaldehyde and the pH dependence of the yield parallels that obtained (14, 15) in 0H(from Ti

Table.

-H 2 0„ or pulse radiolysis) induced reactions.

Summary of photolysis experiments of substrate H O C H ^ C ^ X

(X=NH 2 or OH) in presence of a C o 1 1 1 alkyl showing % of MeCHO formed CoHlalkyl Substrate

pH

7

AdoCbl

8-Me0-AdoCbl

MeCbl

Me(H 2 0) S Cobaloxime

1M H0CH 2 CH 2 0H

2.0

0

- -

11

32

II

7.4

0

- -

8

17

11

11.2

4

- -

36

35

2.0

3

13

34

73

7.4

8

21

17

17

11.2

10

64

46

95 6

9M H0CH 2 CH 2 0H II

"

Neat H0CH 2 CH 2 0H 1M H0CH 2 CH 2 NH 2

- -

0

—

0 0

0

29

25

0

0

2.0

0

- -

II

7.4

0

11

II

11.2

2

- -

7.4

0

- -

EtOCH 9 CH„NH 9

- -

- -

The results are interpreted in terms of an initial hydrogen atom abstraction from substrate by a carbon-centred radical and that the subsequent transformation of this substrate radical into MeCHO is cobalt-independent.

i+76 In the enzyme system, it would seem likely that the 8-position of the coenzyme is "protected" by the protein.

Yurkevich and his co-workers (16)

have also reported that the anaerobic photolysis of AdoCbl and ethylene glycol gave no acetaldehyde (they made a similar statement about MeCbl which conflicts with our observations).

However they also made the in-

teresting observation that when the photolysis was carried out in presence of dihydrolipoic acid amide, 30% of acetaldehyde was obtained. Sulphur-containing intermediates have also been suggested (17, 18) as being involved in B^-nediated rearrangements and Babior (19) in a study of the inactivation of EAL by the sulfhydryl reagent, 5,5'-dithiobis (2-nitro-benzoic acid) has provided further evidence for the importance of -SH groups in the enzymic reaction.

The features depicted in Scheme 1 involving changes in the immediate environment of the cobalt atom make the metal an effective probe for mechanistic studies as recognised in experiments based on e.s.r. (20) which has indicated that a paramagnetic Co(II) species formed at a kinetically competent rate after mixing enzyme with AdoCbl and L-2-aminopropanol.

E.s.r. however provides information only about paramagnetic

species i.e. Co(II), and not Co(I) or Co(III).

Following the significant

role played by Mossbauer spectra in the understanding of the mechanism of biological reactions of iron organometal1ic systems, we examined (21) 57 the

Co Mossbauer emission spectra of hydroxocobalamin, AdoCbl, and the

holo-enzyme ethanolamine ammonia-lyase in frozen aqueous solutions at pH 7.4 and at temperatures between 4-195K.

All 57the spectra proved to be

essentially identical implying that all the

Fe-labelled daughter pro-

ducts were similar, i.e. that the metal-carbon bond was cleaved rapidly on the Mossbauer time-scale following the electron capture. However, another spectroscopic method has been very much more useful in mechanistic studies of the enzyme rearrangement.

This is the method of

stopped-flow rapid wavelength scanning spectrophotometry which involves mixing enzyme and substrate solutions in about 3 ms. and then recording

(+77 spectra over the 345-570 nm range, measuring 800 spectra per second, each spectrum taking 1 ms. with a 0.25 ms. gap between spectra (22).

The

spectra were stored in an ESL data-capture system which enabled the storage of up to 32 spectra at preselected times during the reaction.

In some

of the slower reactions (half-lives of seconds), spectral changes were followed by summing 64 spectra in a single register.

In this case each

averaged spectrum took about 0.15 s to accumulate but the signal-to-noise ratio was improved.

Typical spectra obtained during the reaction with L-

2-aminopropanol as substrate are shown in Figure 2 where Figure 2A is a spectrum recorded in 1 ms and after subtraction of the baseline gives the corrected spectrum in Figure 2C (dotted line) (23).

This spectrum is

similar to the averaged spectrum of Figure 2B, corrected in Figure 2C (solid line) although it has a poorer signal-to-noise ratio (compare Figures 2A and 2B). The transient phases of the reactions were dependent on the order of mixing of reactants.

When enzyme was placed in one syringe of the stopped-

flow apparatus and coenzyme and substrate in the other, the cob(II)alamin intermediate formed slowly (first-order rate constants of ca 5 s L-2-aminopropanol as substrate and ca 2 s 3).

with

with 2-aminoethanol) (Figure

When the enzyme and coenzyme were placed in one syringe and the

substrate in the other, the formation of the cob(II)alamin occurred much -1

more rapidly (90 s ethanol).

-1

with L-2-aminopropanol and >300 s

with 2-amino-

The binding of the adenosylcobalamin to the enzyme protein is

thus followed by a slow change in the conformation of the enzyme molecule to give a catalytically active species (there is no change in the spectrum of the coenzyme concommitant with this process).

The formation

of active holo-enzyme is slow on the time scale of subsequent catalytic steps particularly when 2-aminoethanol is the substrate. The steady-state and post-steady-state spectra gave results which for the substrate L-2-aminopropanol are exemplified by Figure 4.

From the time

courses of Figure 4B it can be seen that the duration of the steady-state phase of the reaction was about 60 s.

Assuming that the substrate (2 mM)

478

I

T

| 0 05 j


300 s ) g r e a t e r than the o v e r a l l k c a t value (140 s ) and has a l i f e time c o n s i s t e n t with t h a t c a l c u l a t e d from the k . v a l u e .

F i g u r e 3. S p e c t r a l changes d u r i n g the r e a c t i o n of ethanolamine ammonial y a s e with AdoCbl as coenzyme and L-2-aminopropanol as s u b s t r a t e and a comparison with spectra of known cobalamin d e r i v a t i v e s . (A) Spectra r e corded a t the i n d i c a t e d times a f t e r mixing enzyme (E) in s y r i n g e 1 ( f i n a l a c t i v e s i t e c o n c e n t r a t i o n in the r e a c t i o n cuvette 14 yM in a c t i v e s i t e s ) with a s o l u t i o n of AdoCbl and L-2-aminopropanol ( P r ) in s y r i n g e 2 ( f i n a l c o n c e n t r a t i o n s 12 yM r e s p e c t i v e l y ) . Only 3 out of the 32 recorded 1-ms s p e c t r a are shown. C o n d i t i o n s were as f o r F i g u r e 2. (B) Logarithmic f i r s t - o r d e r p l o t s of the time c o u r s e s of the r e a c t i o n shown in (A) at 367 nm (A ) , 401 nm (0) and 556 nm ( • ) . In the e x p r e s s i o n g i v e n in the o r d i n a t e a^ r e p r e s e n t s the amplitude of the t o t a l absorbance change at a g i v e n wavelength and x the amplitude a t any time t:. ( C ' ) Spectra o f : cob(II)alamin ( ), cob(I)alamin ( ) and hydroxo cobalamin ( ) (C11) Spectra of ' b a s e - o n ' c o b ( I I ) a l a m i n ( ), 'base-off' c o b ( I I I ) a l a min ( ) , ' b a s e - o n ' c o b ( I I I ) a l a m i n (AdoCbl) ( ) and ' b a s e - o f f ' cob(II)alamin ( ).

I* BO

FiArtoChl

I0

A

Pr

B

I

1 0.05

T

350

400

450

500

Wavelength (rim)

550

Figure 4. The reaction of holoenzyme with L-2-aminopropanol as substrate. (A) Spectra recorded at the indicated times a f t e r mixing enzyme and AdoCbl (syringe 1) with L-2-aminopropanol (Pr) (syringe 2). The f i n a l concentrations in the reaction cuvette were 1 4 y M enzyme active s i t e s , 12 yM coenzyme ( i . e . 12 yM holoenzyme) and 2 mM substrate. The other conditions were as described in Figure 2. Each spectrum i s the average of 64 spectra and was recorded in 0.15 s. (B) Time course of absorbance charges during the reaction described in (A) at 360 nm (0) and 511 nm ( • ) . The spectra of the intermediates formed in reactions with both s u b s t r a t e s , the rates of t h e i r formation and t h e i r l i f e t i m e s allowed a detailed kinet i c a n a l y s i s of the reactions in terms of a three-step mechanism (Scheme 2) i n v o l v i n g binding of the substrate, c o b ( I I ) a l a m i n formation ( k ^ step) and c o b ( I I ) a l a m i n breakdown (J
O ö CU

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cd cd D X -rj X E O - H CU N Q ß i D -

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M

I—I H H H H > H h-II—I H H > l-H •—I HH X H H H H > > > > i - H t - H i - H > > > > i - i x x x x x ; x ? < ? < ?
2 a n d the k e t o a c i d so no m a t t e r

which

c o m p o u n d is o r i g i n a l l y l a b e l e d , the o t h e r a q u i r e s

radioacti-

vity' a n d at the s a m e time as a c o n s e q u e n c e of the

exchange

the s p e c i f i c a c t i v i t y of the o r i g i n a l l a b e14 led compound

de-

creases.

source

It w a s o b s e r v e d

(Fig. 1), w h e n

C 0 2 w a s the

of the label the s p e c i f i c a c t i v i t y of the a c e t a t e w a s low at

533

K

[ K C]cxKetc AC

C02

50.000 _ 40,000 § 30,000

3.

I 20.000 -a 10,000

4

Fig. 1:

8

12

min

i

8

12

Evidence that the carboxylation of the methyl corrinoid is by transcarboxylation and not by fixation of CC>2 (From Schulman et al. (15)).

first and increased with time. The increase accompanied the increase in specific activity of the a-ketobutyrate, which 14 became radioactive because of the exchange of COg with the carboxyl group of the a-ketoglutarate. when

On the other hand

a-ketobutyrate was the source of the label the

specific activity of the acetate was high at first and decreased in specific activity along with the decrease in the specific activity of the a-ketobutyrate.

The latter de-

creased because of the exchange with the cold COg.

Clearly

the specific activity of the acetate is linked to the specific activity of the a-ketobutyrate.

In other experiments,

the rigid exclusion of CO„ was found to have no effect on the 14 14 rate of conversion of CHg-THF and pyruvate to CHgCOOH. Thus, the evidence is clear that C0 2 is not a direct source of the carboxyl of acetate. The discovery that C0 2 is not the direct source of the carbboxyl required modification of the concept of the fermentation as illustrated in Fig. 2. The glucose is converted to 2 pyru14 vates and in the presence of C0 o the pyruvate rapidly 14 aquires C0 2 in its carboxyl group due to the exchange reaction.

The mechanism of the exchange is unknown and this is

indicated by enclosing the 2-carbon compound in brackets. One of the pyruvates is converted to acetate and C0 9 .

The C0 9 is

53k

CO,

GLUCOSE—•2CH,COCOOHI4 2CH,CO 3 < COOHl

CM3COOH

[C2](Exchange)


, 6 4 5

M.

U.S.A.

(1952) (1955)

5.

W o o d , H . G . : J. B i o l . C h e m . 1 9 9 , 519

6.

P o s t o n , J.M., K u r a t o m i , K., S t a d t m a n , E . R . : A n n . N . Y . A c a d . Sei. 112, 804 (1964)

(1952)

7.

T h a u e r , R . K . : F E B S L e t t . 27, 111

8.

A n d r e e s e n , J.R., L j u n g d a h l , L . G . : J. B a c t e r i o l . (1974)

9.

Ljungdahl, L., Brewer, J.M., Neece, S.H., Fairwell, J. B i o l . Chem. 245, 4791 (1970)

(1972) 120, 6 T.:

10. O ' B r i e n , W . E . , B r e w e r , J . M . , L j u n g d a h l , L . G . : i n E x p e r i e n t i a S u p p l e m e n t u m 26, E n z y m e s a n d P r o t e i n s f r o m T h e r m o p h y l i c M i c r o o r g a n i s m s : P r o c e e d i n g s of the I n t e r n a t i o n a l S y m p o s i u m , Z u r i c h (1976) (Zuber, H. e d . ) , B i r k h ä u s e r - V e r l a g , B a s e l , pp 249

538 11.

Andreesen, V.R., Shaup, A., Neuranter, C., Brown, A., Ljungdahl, L.G.: J. Bacteriol. 114, 743 (1973)

12.

Parker, D.J., Wu, T.-F., Wood, H.G.: J. Bacteriol. 108, 770 (1971)

13.

Ghambeer, R.K., Wood, H.G., Schulman, M., Ljungdahl, L.G.: Arch. Biochem. Biophys. 143, 471 (1971)

14.

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Schulman, M., Ghambeer, R.K., Ljungdahl, L.G., Wood, H.G.: J. Biol. Chem. 248, 6255 (1973)

16.

Welty, F.K., Wood, H.G.: J. Biol. Chem. 253, 5832 (1978)

17.

Parker, D.J., Wood, H.G., Ghambeer, R.K., Ljungdahl, L.G.: Biochemistry 11, 3074 (1972)

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MECHANISMS FOR B-, ,-DEPENDENT METHYLATION

J.M. Wood and Y.-T. Fanchiang Gray Freshwater Biological Institute, Department of Biochemistry, College of Biological Sciences, University of Minnesota, P.O. Box 100, Navarre, Minnesota, 55392, USA.

Introduction

At the turn of the century Gosio observed that an unusual gas was evolved from arsenical wallpaper containing the pigments Scheele's green and Paris green (1).

Thirty years later Challenger showed that Gosio's gas

was pure trimethylarsine

(2).

Challenger's discovery provided us w i t h

the first example of how biological systems possess the capability for synthesizing very toxic organo-arsenic compounds from less toxic inorganic substrates (3,4).

In 1968 w e discovered that mercuric salts could b e

biomethylated to give methylmercury and dimethylmercury as products

(5).

Methylcobalamin was shown to be the methylating agent in these reactions. Since this initial discovery a great deal of research has been done on the biomethylation of elements other than mercury

(6-10).

Methyl-transfer

in biological systems naturally depends on the co-enzymes w h i c h are available to perform this function.

Three co-enzymes have been found which

are capable of the transfer of methyl groups:

(1) methylcorrinoid

derivatives, (2) S-adenosylmethionine, and (3) N5 -methyltetrahydrofolate derivatives. For these three coenzymes only methylcorrinoid derivatives are capable of transferring the methyl-group as a carbanion (CH3 ). basis of charge, CH3 n

metal ions (M ^) .

Clearly, on the

is most likely to react w i t h positively

charged

S-adenosylmethionine and N-'-methyltetrahydrofolate

derivatives transfer methyl-groups as carbonium ions (CH^"1") , and so it is unlikely that this CHg"*" species would be involved in transfer to a positively charged metal ion.

© 1979 Walter de Gruyter & Co., Berlin • N e w York Vitamin B ]2

5(+D

Once synthesized, these methylated metals are Invariably more toxic than their inorganic substrates.

This toxicity is probably due to the n o n -

polar nature of many organometallic compounds w h i c h allows them to diffuse rapidly into and through cell membranes.

Dynamic aspects of these methyl-

ation reactions are of critical importance, because even though most methylated metals are thermodynamically unstable in water, many of them are kinetically stable.

In fact, it is well known that metals which are

lower in their periodic groups form metal-alkyls which are kinetically more stable.

For example, mercury, platinum and possibly lead offer

potentially stable systems, whereas palladium, chromium and cadmium do not. In this symposium lecture w e review the different mechanisms for dependent methyl-transfer to a selected group of toxic elements.

Based

on both structural and kinetic studies, six different mechanisms have been formulated which lead to methyl-transfer from methylcobalamin.

Alternate Mechanisms for Co-C Bond Cleavage

Methyl-transfer from methylcobalamin requires cleavage of the Co-C bond. This bond can break under different conditions to give a carbanion (CH3 -1-

a radical (CHg") or a carbonium ion (CHj ) .

),

Figure 1 presents six

alternate reaction pathways which lead to the transfer of the methylgroup by each alternative mechanism. Reaction 1 This pathway involves a single electron oxidation of methylcobalamin by "outer sphere" electron acceptors.

The oxidized methylcobalamin product

is extremely labile and produces a methyl radical and aquocobalamin. Reaction 2 This reaction involves heterolytic cleavage of the Co-C bond w i t h the transfer of a carbanion to the attacking metal ion. Reaction 3 This reaction has been described as a "Redox-Switch" mechanism.

The metal

5U 1

r

nr

>Co
co