Bioorganic Chemistry [1 ed.] 9789350431702, 9788184888355

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BIOORGANIC CHEMISTRY

Dr. G.K. CHATWAL M.Sc. Ph.D. Formerly, Reader in Chemistry OVAL SINGH COLLEGE (University of Delhi) New Delhi-110003.

Edited by

MADHU ARORA

Keil "Himalaya GpublishingGHouse [J [J

MUMBAI [J DELHI [J NAG PUR [J BANGLURU [J HYOERABAD LUCKNOW [J AHMEDABAD [J ERNAKULAM [J BHUBANESWAR

[J [J

CHENNAI [J PUNE KOLKATA [J INDORE

©

Author Stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording and/or otherwise without the prior written permission of the author.

First Edition: 2010

ISBN : 978-81-84888-35-5 Published by

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Contents Chapter 1 Bioorganic Chemistry

1.1 - 1.23

Chapter 2 Enzymes

2.1 - 2.87

Chapter 3 Mechanism of Enzyme Action

3.1 - 3.12

Chapter 4 Kinds of Reactions Catalysed by Enzymes

4.1 - 4.14

Chapter 5 Coenzyme Chemistry

5.1 - 5.19

Chapter 6 Enzyme Models

6.1 - 6.68

Chapter 7 Biotechnological Applications of Enzymes

7.1 - 7.72

"This page is Intentionally Left Blank"

CHAPTEI

BIOORGANIC CHEMISTRY II 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 I I II

Introduction Bioorganic chemistry is a rapidly growing scientific discipline which combines organic chemistry and biochemistry. While biochemistry aims at understanding biological processes using chemistry, bioorganic chemistry attempts to expand organic-chemical researches (that is, structures, synthesis, and kinetics) toward biology. When investigating metalloenzymes and cofactors, bioorganic chemistry overlaps bioinorganic chemistry. Biophysical organic chemistry is a term used when attempting to describe intimate details of molecular recognition by bioorganic chemistry. Bioorganic chemistry is that branch of life science, which deals with the study of biological processes using chemical methods. Bioorganic chemistry is mainly concerned with the study of various organic processes, such as oxidation, reduction, cyclisation dissociation, transport through biomembranes, reaction catalysts via biological catalysts (enzymes), etc. These organic processes are running in bio-systems. Most important of all these organic processes are enzyme catalysed reactions which play vital role in the maintenance of life processes on the earth. Bioorganic chemistry is of utmost importance in our present life style because different food stuffs (e.g., bread from fermented douge; cheese, curd and brewed products), medicinal syrups and various drinks are products of bioorganic reactions. Fermentation is a bioorganic process occurring in presence of bio-catalyst from yeast; pectinases are usually added to canned fruit juices and to wine because as they hydrolyse the pectin making the juice or wine clear; enzyme invertase finds used in the manufacture of chocolate. These are few among myriads of modern bio-organic applications. Bioorganic chemistry is the application of the principles and the tools of organic chemistry to the understanding of biological process. Such an understanding is often achieved with the aid of molecular models chemically synthesised in the laboratory. This permits a sorting out of the many variable parameters simultaneously operative within the biological system. An example is how does a biological membrane work? One constructs a simple model of known compositions and studies a single behavior, such as an ion transport property .

.

What are the tools required for bioorganic model studies? Organic and physical organic chemical principles will provide the best opportunities for model building(1.1)

1.2

Bioorganic Chemistry

modeling molecular events that form the basis of life. Many of those results have been found out to be wonderful tools for the discovery and characterization of specific molecular events in living systems. Think of the development of antibiotics, specific alkaloids, and the design of new drugs for the medicine of today and tomorrow. All living processes need energy, which is obtained by carrying out chemical reactions inside cells. These biochemical processes are based on chemical dynamics and involve reductions and oxidations. Biological oxidations are thus the main source of energy for deriving a number of endergonic biological transformations. Many reactions involve combustion of foods like sugars and lipids to produce energy which finds use for a variety of essential functions like growth, replication, maintenance, muscular work, and heat production. These transformations are also related to oxygen uptake; breathing is a biochemical process by which molecular oxygen gets reduced to water. Throughout these pathways, energy is stored in the form of adenosine triphosphate (ATP) , which is known as an energy-rich compound known as the universal product of energetic transactions. A portion ofthe energy from the combustion engine in the cell finds use to perpetuate the machine. The machine is composed of structural components that must be replicated. Ordinary combustion yields only heat plus some visible light and waste. Biological combustions, however, yield some heat but a large portion of the energy is used to drive a "molecular engine" that synthesizes copies of itself and that does mechanical work as well. As these transformations take place at low temperature (body temperature, 37°C) and in aqueous media, catalysts are necessary for smooth or rapid energy release and transfer. Thus, apart from structural components, molecular catalysts are needed. These catalysts should be highly efficient (a minimum of waste) and highly specific when precise patterns are to be produced. If bondbreaking and bond-forming reactions are to be carried out on a specific starting material, then a suitable specific catalyst capable of recognizing the substrate have to be "constructed" around that substrate. This fundamental question is posed by all biochemical phenomena, a substrate molecule and the specific reaction it must undergo must be translated into another structure of much higher order whose information content perfectly matches the specifically planned chemical transformation. Only large macromolecules may be able to carry enough molecular information both from the point of view of substrate recognition and thermodynamic efficiency of the transformation. These macromolecules are proteins. They should be extremely versatile in the physicochemical sense because innumerable substrates of widely divergent chemical and physical properties must all be handled by proteins. Therefore, protein composition must be amenable to wide variations in order that different substrates may be recognized and handled. Some proteins will even require adjuncts (nonprotein parts) to assist in recognition and transformation. These cofactors are known as coenzymes. One can therefore predict that protein catalysts or enzymes must possess a high degree of order and organization. Further, a minimum size would be essential for all the information to be contained. These ordered biopolymers, which permit the combustion engine to work and to replicate itself, must also be replicated exactly once a perfect translation of substrate

Bioorganic Chemistry

1.3

structure into a specific function has been established. Hence, the molecular information in the proteins (enzymes) must be safely stored into stable, relatively static language. This is where the nucleic acids enter into the picture. As a result, another translation phenomenon involves protein information content written into a linear molecular language which can be copied and distributed to other cells. The best way to vary at will the information content of a macromolecule is to use some sort of backbone and to peg on it various arrays of side chains. Each side chain may carry well-defined information regarding interactions between themselves or with a specific substrate so as to perform specific bond-making or breaking functions. Nucleic acid-protein interactions should also be mentioned due to their fundamental importance in the evolution of the genetic code. The backbone just described is a polyamide and the pegs are the amino acid side chains. Why polyamide? Because it has the capacity of "freezing" the biopolymer backbone into precise three-dimensional patterns. Flexibility is also achieved and is of considerable importance for conformational "breathing" effects to occur. A substrate can therefore get transformed in terms of protein conformation imprints and, finally, mechanical energy might also be translocated. The large variety of organic structures known provide an infinite number of structural and functional properties to a protein. Using water as the translating medium, one can go from nonpolar (structured or nonstructured) to polar (hydrogen-bonded) to ionic (solvated) amino acids; from aromatic to aliphatics; from reducible to oxidizable groups. Thus, almost the entire encyclopedia of chemical organic reactions can be coded on a polypeptide backbone and tertiary structure. Finally, since all amino acids present share of L (or S) configuration, one can realize that chirality is necessary for order to exist.

Proximity Effects in Organic Chemistry The proximity of reactive groups in a chemical transformation permits bond polarization, causing generally in acceleration in the rate of the reaction. In nature this is normally attained by a well-defined alignment of specific amino acid side chains at the active site of an enzyme. Study of organic reactions is helpful to construct proper biomodels of enzymatic reactions and open a field of intensive research : medicinal chemistry through rational drug design.

•

The first example involves the hydrolysis of a glucoside bond. o-Carboxyphenyl ~D-glucoside gets hydrolyzed at a rate 10 4 faster than the corresponding p-carboxyphenyl analog. Hence, the carboxylate group in the ortho position must "participate" or be involved in the hydrolysis.

Bioorganic Chemistry

1.4

HO

~ 0

+

HO

HO

OH

OH

HOY'll

HOy0 o

o-Carboxyphenyl poD-glucoside This ascertains the fact that the proper positioning of a group (electrophilic or nucleophilic) might accelerate the rate of a reaction. There is thus an analogy to be made with the active site of an enzyme m,e lysozyme. Of course, the nature of the leaving group is also important in describing the properties. Also, solvation effects can be of paramount importance for the course of the transformation, especially in the transition state. Reactions of this type are known as assisted hydrolysis and take place by an intramolecular displacement mechanism; steric factors may retard the reactions. We will now look at another example: 2, 2'-tolanocarboxylic acid in ethanol is converted to 3-(2-carboxybenzilidene) phthalide. The rate of the reaction has been 10 4 faster than with the corresponding 2-tolancarboxylic or 2, 4' -tolancarboxylic acid. One carboxyl group behaves as a general acid catalyst by a mechanism called complementary bifunctional catalysis. HOOC

ErOR~

C?:tCH~

)

o 2, 2' -Tolan carboxylic acid

3, (2-Carboxybenzilidene) phthalide

The ester function of 4-(4'-imidazolyl) butanoic phenyl ester (1) gets hydrolyzed much faster than the corresponding n-butanoic phenyl ester. If there is a p-nitro group on the aryl residue, the rate of hydrolysis has been even faster at neutral pH. As expected, the presence of a better leaving group further accelerates the rate of reaction. This hydrolysis involves the formulation of a tetrahedral intermediated.

,Q Qfo~

H~

(1)

-PhOR .

O-Ph

(2)

1.5

Bioorganic Chemistry

The imidazole group behaves as a nucleophilic catalyst in this two-step conversion, and its proximity to the ester function and the formation of a cyclic intermediate have been the factors responsible for the rate enhancement observed. The involvement of an i)lli.dazole group in the hydrolysis of an ester may denote the simplest model of hydrolytic enzymes.

(6)

In a different domain, amide bond hydrolyses can also get accelerated. An example is the following where the reaction gets catalyzed by a pyridine ring. The first step involves the rate-limiting step of the reaction (slow reaction) leading to an acyl pyridinium intermediate (6), reminiscent of a covalent acyl-enzyme intermediate found in many enzymatic mechanisms. This intermediate is then rapidly trapped by water. The example taken from the steroid field demonstrates the importance of a rigid framework. The solvolysis of acetates (8) and (9) in CHsOHlEtsN depicted a marked preference for the molecule having a ~OH group at carbon 5 where the rate of hydrolysis has been 300 times faster.

H

AcO

OH (9)

(8)

cis junction The explanation for such a behaviour becomes apparent if the molecule is drawn in three dimensions (10). The rigidity of the steroid skeleton thus helps in bringing the two functions into proper orientation where catalysis combining one intramolecular and one intermolecular catalyst occurs. The proximal hydroxyl group can cooperate in the

Bioorganic Chemistry

1.6

hydrolysis by hydrogen bonding, and the carbonyl function of the ester becomes a better electrophilic center for the solvent molecules. In this mechanism, one can ·perceive a general acid-base catalysis of ester solvolysis.

/0 H

~.

0········0

y.-......... /H

NEt3

·

CH '0

I

3

CHa (10)

These simple examples show that many of the basic active site chemistry of enzymes can be reproduced with simple organic models in the absence of proteins. The role of the latter is of substrate recognition and orientation and the chemistry is al~o performed by cofactors (coenzymes) that also have to be specifically recognized by the protein or enzyme. Molecular Adaptation Besides proximity effects there are other factors which should be taken into account in the design of biomodels. For instance in 1950 Friedman introduced the concept of bioisosteric groups. In its broadest sense, the term refers to chemical groups that bear some resemblance in molecular size and shape and as a consequence can compete for the same biological tar-get. This concept finds important application in molecular pharmacology, especially in the design of new drugs through the method of variation, or molecular modification. There are some pharmacological examples that will illustrate the principle. The two neurotransmitters, acetylcholine (A) anq carbachol (B), have similar muscarinic action. $

N(CHa)3 I I I I

(A) acetylcholine

$

N(CH 3)3

: : ll I

I

I

I I

$

(B) carbachol

HO

HaC

I

~(CHah

I I I

I I ~

_

~

0.44nm

I

(C) muscarine

1.7

Bioorganic Chemistry

The shaded area denotes the bioisosteric equivalence. Muscarine (C) is an alkaloid that inhibits the action of acetylcholine. It is found, for example, in Amanita muscaria (fly agaric) and other poisonous mushrooms. Its structure implies that, in order to block the action of acetylcholine on receptors of smooth muscles and glandular cells, it must bind in a similar fashion. 5-Fluorocytosine is an analog of cytosine which is commonly used as an antibiotic against bacterial infections. One serious problem in drug design is to develop a therapy which will not harm the patient's tissues but will destroy the infecting cells or bacteria. A novel approach is to "disguise" the drug so that it is chemically modified to gain entry and kill invading microorganisms without affecting normal tissues. The approach involves exploiting a feature which is common to many microorganisms: peptide transport. Henet'. the amino function of compound is chemically joined to a small peptide. This pepl ide is having D-amino acids and hence avoids hydrolysis by common human enzymes and entry into human tissues. However, the drug-bearing peptide can sneak into the bacterial cell. It then gets metabolized to liberate the active antifungal drug which kills only the invading cell. This is the type of research that the group of W.D. Kingsbury is undertaking at Smith Kline and French Laboratories. This principle of using peptides to carry drugs has been applicable to many different disease-causing organisms.

H 5·Fluorocytosine

;:5H

cytosine

In a similar way, 1-~-D-2'-deoxyribofuranosyl-5-iodo-uracil has been an antagonist of 1-~-D-2'-deoxyribofuranosyl thymine, or thymidine (2). That is, it is able to antagonize or prevent the action of the latter in biological systems, though it may not carry out the same function. Such an altered metabolite is also known as an antimetabolite.

HN

O~

HO~ OH (1)

(2)

Another example of molecular modification has been the synthetic nucleoside adenosine arabinoside. This compound is having a pronounced antiviral activity against herpes virus and is therefore mainly used in modern chemotherapy. The analogy with deoxyadenosine, a normal component of DNA, has been striking. Except for the presence of a hydroxyl group at the 2' -sugar position, the two molecules

Bioorganic Chemistry

1.8

have been identical. Compared to the ribose ring found in the RNA molecule, it is having the inverse or epimeric configuration and hence belongs to the arabinose series. Interestingly, a simple inversion of configuration at C-2' confers antiviral properties. Its mechanism of action has been well studied and it does act, after phosphorylation, as a potent inhibitor of DNA synthesis. In a similar way cytosine arabinoside ~as been the most effective drug for acute myeloblastic leukemia. NH2

(:6 N

HO

HO

antivu:al antibiotic

deoxyadenosine

An interesting fact was the finding that this antiviral antibiotic is in fact produced by bacterium called Streptomyces antibioticus. This permits the production by fermentation of large quantities of this active principle.

a

Even more spectacular is the fact that the acyclonucleotide analog Acyclovir has now been licensed for clinical use for the treatment of herpes simplex. This potent antiherpes drug inhibits nucleoside phosphorylating enzymes and this affects growth and replication of cells. The knowledge of the mode of action of such a "target enzyme" inhibitor is fundamental for the design of new drugs for cancer chemotherapy.

o

(DH

HO~O~

N

NH2

Acyclovir

For maximal activity, the open chain molecule adopts a conformation close enough to mimic the furanose ring of the natural nucleosides. An X-ray work has confirmed such a molecular arrangement. Many organophosphonates are synthesized as bioisosteric. analog for biochemically important non-nucleoside and nucleoside phosphates. For example, the S-enantiomer of 3, 4-dihydroxybutyl-l-phosphonic acid (DHBP) is synthesized as the isosteric analog of . sn-glycerol 3-phosphate. The former material has been bacteriostatic at low concentrations to certain strains of E. coli and B. subtilus. As sn-glycerol 3-phosphate has been the backbone of phospholipids (an imp.o rtant cell membrane constituent) and has been able to enter into lipid metabolism and the glycolytic pathway, it has been sensitive to a number of enzyme-mediated processes. The phosphonic acid can participate, but only up to a point, in these cellular reactions. For example, it cannot be hydrolyzed to release glycerol and inorganic phosphate. Of course, the R-enantiomer lacks biological activity.

Bioorganic Chemistry

1.9

H ;; r HOiC-CH2 HO CH z S-DHBP

s1!-gycer o1 3-phosphate

The presence of a halogen atom on a molecule sometimes gives rise to interesting properties_ For example, substitution of the 9a position by a halogen in cortisone does increase the activity of the hormone by prolonging the half-life of the drug. The activity enhances in the following order : X = I > Br > CI > F > H. These cortisone analogs are used in the diagnosis and treatment of a variety of disorders of adrenal function and as anti-inflammatory agents.

OH

Cortisone

As another example, the normal thyroid gland has been responsible for the synthesis and release of an unusual amino acid called thyroxine . This hormone is able to regulate the rate of cellular oxidative processes. The presence of the bulky atoms of iodine disallows free rotation around the ether bond and forces the planes of aromatic rings to remain perpendicular to each other. However, many halogen-free derivatives still possess significant thyromimetic activity. As there is no absolute requirement for iodine or any halogen for thyroid hormone activity in amphibians and mammals, it is likely that the primary role of iodine is rather to provide appropriate spacial constraints on semirigid biosynthetic amino acid precursors and in a way tends to facilitate the biosynthesis of the hormone itself in the thyroid gland. Thus it is an example of a conformational control at the biosynthetic level.

.

The presence of alkyl groups or chains can also affect the biological activity of a substrate or a drug. An interesting case is the antimalarial compounds derived from 6-methoxy-8~aminoquinoline (primaquine analog). The activity is higher in compounds

Bioorganic Chemistry

1.10

in which n is an even number is the range of n = 2 to 7. So the proper fit of the side chain on a receptor site or protein is somehow controlled by the size and shape of the side chain.

H,CO

W HN "

j C Ha (CH 2)'I- N '---CH3

primaquine drug

Finally, we should mention about molecular adaptation at the conformational leveL The development of a certain "designer drug" is a case history. In the early 1980s, the sudden appearance of parkinsonian symptoms in young drug abusers was very unusual because this disease affects mainly persons over fifty years old. How did this happen? The origin of this "frozen addict" behavior was finally traced back to a "new heroin" type agent. The compound had been manufactured in a clandestine laboratory in San Jose, California, and was termed as a designer drug. Meperidine, or Demerol, is a well-known analgesic. It is less potent than morphine but its action is shorter and it is very addictive. Knowing this, the clandestine chemist tried to synthesize a similar compound, but easier to make, that turned out to be also very active. It was MPPP.

o~

morphine supel'imposible to morphine structure

meperidine (Demerol)

accessibly simpler

~J o MPPP l-methylpropionoxyphenyl-piperidine

However, when heated to evaporate the solvent and when the pH is not controlled and too low, the molecule possesses a tendency to hydrolyze and dehydrate to yield a

Bioorganic Chemistry

1.11

by-product, MPTP. Unfortunately, this unnoticed impurity in the faulty batch was the precursor to the key compound responsible for the unexpected side effects of the designer drug, originally conceived to be an readily accessible and simpler analgesic. In reality, the neutral MPTP substance gets readily oxidized by a monoamine oxidase enzyme (MAO) in the brain to an ionic MPP+ molecule which is a neurotoxic pyridinium metabolite. Similar pyridinium salts currently find use as herbicides. Admittedly, the incidence of Parkinson's disease is distinctly higher in rural regions as compared to urban ones since similar chemicals find use in agriculture. This example illustrates well how, by accident, a faulty batch of an assumable good drug gave rise to unexpected parkinsonism in young drug abusers. This lays emphasis on the point that proper conformation can provide (sometimes unexpectedly) a compound having very unusual therapeutic properties where analogs can be exploited.

_

IfD

MPPP~

becomes toxic by passing through ~ the blood-brain ...,....... barrier

MPTP

MPP+

I-methyl-4-phenyl-l, 2, 3, 6-tetrahydropyridine

I-methyl-4-phellyl-pyridinium (a lVlAO met abolite)

Besides the steric and external shell factors inductive and resonance contributions are also important. All these factors must be taken into account in the planning of any molecular biomodel system that will hopefully have the anticipated property. Thus, small but subtle changes on a biomolecule can confer to the new product large and important new properties.

Molecular Recognition and Supramolecular Level By the end of the present century, most of research chemists will use some aspects of molecular recognition in catalysis, affinity chromatography and controlled drug release. Molecular recognition means not only more binding but also selection and possibly specific function. As such molecular recognition represents a binding mode with a purpose as does a real biological receptor. At this point, it could be instructive to draw a global picture of the methodology used to construct bioorganic models of biological systems. The general strategy is outlined in Fig. 1. Basically in this strategy, two distinct but interconnected chemical levels are involved. On one level, a bioorganic receptor is constructed by involving classical organic synthesis. Such a receptor will find use to recognise and interact specially with a given substrate and reach the second level of molecular interaction, the supramolecular level.

Bioorganic Chemistry

1.12

In the development, all resources of molecular chemistry are needed. As a result, the efficiency of interaction and activity of the biomodel compound would be limited only by the degree of imagination of the bioorganic chemist. The supermolecule may be divided into three distinct areas, each of which is having a specific function and must answer particular needs. This is summarized in Fig. 2. More specifically, molecular recognition in the design of a receptor involves the understanding of many interactions at the molecular level, such as follows : • Structural information. • Functional information. • Organic and inorganic molecular adaptation I}.nd recognition of intermolecular chemical bonds. • The energetic of the process. • The three-dimensional complementarily of molecular shapes, and • The concept of molecular cavity as an architectural principle.

c).

o*--~)+~

( t~ ~

complexation

covalent 'bonds

'N'

chemical synthesis by various methods '-

specific in termolecular bonds

______________ J

~

L~

_________

~

Molecular Level of complexity

Receptor : .. :................:::::::::::::::::::::::.:::::::~ Supermolecule

f.' ort Catalytic&.~~¥M:::::::::::::::::::::::::::::::::::::::::::::j

Ion Tran

Moleeulat~&

Supi~~mlOl~~ De.yice~ C = 0)

3

acyl group (- C- R)

II

o 4

glycosyl (carbohydrate) group

7

phosphate group

In general, the third digit further describes the group transferred. Hence E.C. 2.1.1 enzymes are methyltransferases (transfer -CHs) whereas E.C. 2.1.2 enzymes are hydroxymethyltransferases (transfer -CH 20H) and E.C. 2.1.3 enzymes are carboxyl- or carbamoyl-transferases (transfer - C- OH or

II

- C - NH 2 ).

o"

0

2.8.

Bioorganic Chemistry

Similarly, E.C. 2.4.1 enzymes are hexosyltransferases (transfer hexose units) and E.C. 2.4.2 enzymes are pEmtosyltransferases (transfer pentose units). The exception to this general rule for transferases is where there is transfer of phosphate groups : E.C. 2.7.1 enzymes are phosphotransferases with an alcohol group as acceptor, E.C. 2.7.2 enzymes are phosphotransferases with a carboxyl group as acceptor, E.C. 2.7.3 enzymes are phosphotransferases with a nitrogenous group as acceptor . • Phosphotransferases usually have a trivial name ending in '-kinase'. Some example of transferases are : (S)-2-methyl-3-oxopropanoyl-CoA : Pyruvate carboxyltransferase (E.C. 2.1.3.1) (trivial name: methylmalonyl-CoA carboxyltransferase, formerly transcarboxylase) which catalyses the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate

methylmalonyl-CoA

pyruvate

propionyl-CoA

oxaloacetate

ATP : D-hexose-6-phosphotransferase (E.C. 2.7.1.1.) (trivial name: hexokinase) which catalyses:

D-hexose

D-hexose-6-phosphate

This enzyme will transfer phosphate to a variety of D-hexoses. Main Class 3 : Hydrolases : These enzymes catalyse hydrolytic reactions of the form: A - X + H 20

~

X - OH + HA.

They are classified according to the type of bond hydrolysed. For example Second Digit

Bond Hydrolysed

1

ester

2

glycosidic (linking carbohydrate units)

4

peptide

5

C - N bonds other than pep tides

¥

(- c- N-)

~

The third digit further describes the type of bond hydrolysed. Hence

°II

E.C. 3.1.1. enzymes are carboxylic ester (-C-O-) hydrolases,

Enzymes

2.9

o II

E.C. 3.1.2 enzymes are thiol ester (-C-S-) hydrolases, E.C. 3.1.3 enzymes are phosphoric monoester (-0 -

PO~-) hydrolases,

-0

I II .

E.C. 3.1.4 enzymes are phosphoric diester (-O-P-O-) hydrolases.

o

An example is orthophosphoric monoester phosphohydrolase (E.C. 3.1.3.1) (alkaline phosphatase) catalyses : -0 -0

I II

R-O-P-O- + H.O

~

I

R-OH+ HO-P-O-

II

2

o

o organic phosphate

inorganic phosphate

Alkaline phosphatases are relatively non-specific, and act on a variety of substrates at alkaline pH. The trivial names of hydrolases are recommended to be the only ones to consist simply of the name of the substrate plus '-ase'. Main Class 4 : Lyases : These enzymes are able to catalyse the non-hydrolytic removal of groups from substrates, often leaving double bonds. The second digit in the classification shows the bond broken, for example, Second Digit

Bond Broken

1

C-C C-O C-N C-S

2 3

4

The third digit refers to the type of group removed. Thus, for the C - C lyases : Third Digit

Group Removed

1

carboxyl group (i.e. CO 2)

2

aldehyde group (- CH

3

keto acid group (-C.C0 2-)

= 0)

II

o An example is L-histidine carboxy-lyase (E.C. 4.1.1.22) (trivial name decarboxylase, catalyses: +

CaN2Ha.CH2CH.NH3+ ~ CaN2Ha.CH2.CH2.NHa + CO 2

I

CO 2histidine

histamine

histidine

Bioorganic Chemistry

2.10

It is to be noted that the importance of the hyphen and the extra 'y' in the systematic name, because carboxy-lyase and carboxylase do not mean the same thing: carboxylase simply refers to the involvement of CO 2 in a reaction without being specific. Also classified as lyases are enzymes catalysing reactions whose biochemically important direction is the reverse of the above, i.e. addition across double bonds. These may have the trivial name synthase or, if water is added across the double bond, hydratase, in the example of fumarate hydratase (fumarase); the systematic name of this particular enzyme is (S)-malate hydro-lyase (E.C. 4.2.1.2). Main Class 5 : Isomerases : Enzymes catalysing isomerization reactions can be classified according to the type of reaction involved. For instance, Type of Reaction

Second Digit 1

Racemization or epimerization (inversion at an asymmetric carbon atom)

2

cis-trans isomerization

3

Intramolecular oxidoreductases

4

Intramolecular transfer reaction

The third digit is describing the type of molecule undergoing isomerization. Hence for racemases and epimerases Substrate

Third Digit

Amino acids Hydroxy acids Carbohydrates

1

2 3

An example has been alanine racemase (E.C. 5.1.1.1) which is able to catalyse

L - alanine

~

D - alanine

Main Class 6 : Ligases : These enzymes are able to catalyse the synthesis of new bonds, coupled to the breakdown of ATP or other nucleoside triphosphates. The reactions are of the form given below :

x or

+ Y + ATP

X + Y + ATP

X - Y + ADP + Pi X - Y + AMP + (PP)i

The second digit in the code represents the type of bond synthesized. For instance Second Digit

Bond Synthesized

1

C-o

2

c-s

3

C-N

4

C-C

2.11

Enzymes The third digit further indicates the bond being formed. Hence,

o II

E.C. 6.3.1 ,enzymes have been acid-ammonia ligases (amide, -C-NHz' syntheses) and

o II

E.C. 6.3.2 enzymes have been acid-amino acid ligases (peptide, -C-N-. H . syntheses). Prior to 1984, such enzymes were also known as synthetases. An example has been L-glutamate : ammonia ligase (E.C. 6.3.1.2) trivial name glutamate-ammonia ligase, formerly glutamine synthetase) which is known to catalyse:

L-glutamate

L-glutamine

Structure of Enzymes All enzymes are proteins. Proteins are high molecular weight macromolecules. An enzyme may consist ofa single polypeptide chain, e.g. beef ribonuclease, or an aggregate of polypeptide chains. The polypeptide chain is made up of a number of amino acid units linked by peptide bonds. The sequence and number of the 20 amino acid which make up enzymes varies in different enzymes. This sequence is specific for a particular enzyme and determines the properties of the enzyme. The amino acid sequence and the three dimensional structure have been determined for the enzymes egg-white lysozyme, ribonuclease, carboxypeptidase, chymotrypsin and papain (Fjg. 2.1). Lysozyme hydrolyses links between amino sugars, and is found in saliva and egg white. Ribonuclease degrades RNA into small fragments. Chymotrypsin and trypsin ate proteolytic enzymes found in the pancreas. Chymotrypsinogen, the inactive precursor of chymotrypsin, consists of a single polypeptide chain. Removal of two dipeptides results in the active enzyme chymotrypsin, which consists of three polypeptide chains. Lysozyme, ribonuclease, carboxypeptidase, chymotrypsinogen and papain each consist of a single polypeptide chain which is highly folded into a spherical shape. Trypsinogen consists of two 'polypeptide chains, and chymotrypsin of three chains. The number of amino acid residues is 33 in carbonic anyhydrase, 124 in ribonuclease, 129 in egg-white lysozyme and 249 in chymotrypsinogen and trypsinogen. The polypeptide chain has an amino (-NH2) terminal and a carboxy (-COOH) terminal. Biosynthesis of the enzyme begins at the amino terminal. . The different parts of the polypeptide chain are linked by disulphide (-8-8-) bridges, which are most commonly found between two cysteine amino acids. Disulphide bridges may be within a polypeptide chain (intrachain) or may connect two polypeptide chains (interchain). Egg-white lysozyme and ribonuclease have four disulphide bridges, chymotrypsinogen has give and trypsinogen six.

Bioorganic Chemistry

2.12

Fig. 2.1 : The structure of beef ribonuclease. Note the four disulphide bridges between cysteine-cysteine

Enzyme Action Thert~ is a very close structural relationship between the molecular surface of an enzyme and its substrates. Enzymes are protein molecules with definite surface geometry.

Enzymes

2.13

The functional groups of the enzyme are exactly complementary to those of the substrate. Only particular types of substrate molecules will fit with a given enzyme molecule. For example, substrates A and B will fit into the enzyme E but not substrate C (Fig. 2.2). This is referred to as a 'lock-and-key' mechanism. Thus reactions involving A and B will e speeded up but, not reactions involving C.

Enzyme

Fig. 2.2 : Diagram illustrating how only particular substrates react with an enzyme. The enzyme enters into a chemical combination with the substrate to form an enzyme-substrate complex (Michaelis-Menton hypothesis). E +S

----7

ES

The enzyme-substrate complex then breaks down to give the products of reaction. The enzyme is released and can be used over and over again. ES

----7 . E

+ Products

Activation Energy (Fig. 2.3) Enzymes increase the speed of a chemical reaction. They lower the energy of activation of a reaction, thus enabling it to occur at ordinary physiological temperatures. When reactions proceed from one direction to another they have to overcome an energy barrier called the activation energy. Normally only a small part of the total number of molecules in a compound contain enough energy to react. Application of heat enables a larger proportion of molecules to overcome the activation energy barrier. However, living systems are relatively isothermal, i.e. they do not have large temperature differences. They therefore employ enzymes to activate the molecules of the reacting compounds. When the enzyme combines with the substrate to form the enzyme-substrate complex, the energy level of the substrate is raised, and it reacts faster. Enzymes increase the speed of a chemical reaction thousands of times by bringing about mutual contact of reacting compounds. The coming together of these compounds is no longer a matter of chance, but becomes a certainty.

Bioorganic Chemistry

2.14

"J A .E.

®

Course of reaction Fig. 2.3

Temperature

pH

Fig. 2.4

Fig. 2.5

Fig. 2.3 : Change in activation energy (A.E.). (A) Reaction without enzyme. (B) Reaction with enzyme. Fig. 2.4 : Effect of temperature on the rate of enzymatic reaction. On represents the point where thermal denaturation of the enzyme begins (45-55°C). Fig. 2.5 : Effect of pH on the rate of enzyme reaction. The dashed line indicates optimum pH. pH At optimum pH the activity of enzymes is maximum (Fig. 2.5). For most enzymes the effective pH range is 4-9. Beyond these limits denaturation of enzymes takes place. The optimum pH for pepsin is 2.0 and for trypsin 8.0. Ions Enzyme activity is affected by H ion concentration and other ionic concentrations. Some enzymes require a loosely bound cation such as Mg++.

Effects of pH and Ionic Strength Enzymes are amphoteric molecules containing a large number of acid and basic vary, according to groups situated on their surface. The charges on these groups their acid dissociation constants, .with the pH of their environment (Table 2.1). This will effect the total net charge of the enzymes and the distribution of charge on their exterior surfaces, in addition to the reactivity of the catalytically active groups. These effects are especially important in the neighbourhood of the active sites. Taken together, the changes in charges with pH affect the activity, structural stability and solubility of .the enzyme. .

will

The pKa (defined as -LoglO(Ka» is the pH at which half the groups are ionised. Note the similarity between the Ka of an acid and the Km of an enzyme, which is the substrate concentration at which half the enzyme molecules have bound substrate. By convention, the heat (enthalpy) of ionisation is positive when heat is withdrawn from the surrounding solution (i.e. the reaction is endothermic) by the dissociation of the hydrogen ions.

Enzymes

2.15

Table 2.1 : pKasa and Heat of Ionisationb of the Ionising Groups Commonly Found in Enzymes Group

Usual pKa Range

Carboxyl (C-terminal, glutamic acid, aspartic acid) Ammonia (N-terminal) (lysine) Imidazolyl (histidine) Guanidyl (arginine) Phenolic (tyrosine) Thiol (cysteine)

3-6 7-9 9 - 11

5- 8 11 - 13

Approximate Charge at pH 7

Heats of Ionisation (kJ mole-i)

- 1.0

±5

+ + + +

1.0 1.0 0.5 1.0

9 - 12

0.0

8 - 11

0.0

+ + + + + +

45 45 30 50 25 25

There will be a pH, characteristic of each enzyme, at which the net charge on the molecule is zero. This is called the isoelectric point (PI), at which the enzyme generally has minimum solubility in aqueous solutions. In a similar manner to the effect on enzymes, the charge and charge distribution on the substrate(s), product(s) and coenzymes (where applicable) will also be affected by pH changes. Increasing hydrogen ion concentration will, additionally, increase the successful competition of hydrogen ions for any metal cationic binding sites on the enzyme, reducing the bound metal cation concentration. Decreasing hydrogen ion concentration, on the other hand, leads to increasing hydroxyl ion concentration which compete against the enzymes' ligands for divalent and trivalent cations causing their conversion to hydroxides and, at high hydroxyl concentrations, their complete removal from the enzyme. The temperature also has a marked effect on ionisations, the extent of which depends on the heats of ionisation of the particular groups concerned (Table 2.2). The relationship between the change in the pKa and the change in temperature is given by a derivative of the Gibbs-Helmholtz equation.

d (pKa) dT

-8H

=----::-

2.303RT 2

(1)

where T is the absolute temperature (K), R is the gas law constant (8.314 J M-l K-l), 8H is the heat of ionisation and the numeric constant (2.303) is the natural logarithm of 10, as pKa's are based on logarithms with base 10. This variation is sufficient to shift the pI of enzymes by up to one unit towards lower pH on increasing the temperature by 50°C. These charge variations, plus any consequent structural alterations, may be reflected in changes in the binding of the substrate, the catalytic efficiency and the amount of active enzyme. Both V max and Km will be affected due to the resultant modifications to the kinetic rate constants k+l' k_l and k cat (k+2 in the Michaelis-Menten mechanism), and the variation in the concentration of active enzyme. The effect of pH on the V max of an enzyme catalysed reaction may be explained using the, generally true, assumption that only one charged form of the enzyme is optimally catalytic and therefore the maximum concentration of the enzyme-substrate intermediate cannot be greater than the concentration of this species. In simple terms, assume EH- is the only active form of the enzyme,

2.16

Bioorga.nic ChellHlWY

EH- + S ~~k±:::J=~ EH-S _---'k+=2_~) P k_l

(1.8)

There concentration of EH- is determined by the two dissociations (1.9)

EH

(1.10)

J

[EH- o [H+ [EH 2]0

with

J

(1.13)

[E 2 - Jo [H+ J

and

(1.14)

J

[EH- o (1.15)

therefore

[El.

~ [EW]. ([;:IJ+ 1 + [:-:Jl

(1.16)

As the rate of reaction is given by k+2 [EH-S] and this is maximal when [EH-S] is maximal (i.e. when [EH-S] = [EH]o) :

Vmax =

k

[ H +2 E

-J

k+2 [E]o

= ([ +

J

-H- + 1 +Ka2 -Kal

[H+

1

J

(1.17)

The V max will be greatest when (1.18)

therefore

(1.19)

This derivation hm; involved a number of simplifications on the real situation; it ignores the effect of the ionisation of substrates, products and enzyme-substrate complexes and it assumes EH- is a single ionis9d species when it may contain a mixture of differently ionised groups but with identical overall charge, although the process of binding substrate will tend to fix the required ionic species. It does, however, produce a variation of maximum rate with pH which gives the commonly encountered 'bellshaped' curve (Fig. 2.6). Where the actual reaction scheme is more complex, there may be a more complex relationship between V max and pH. In partic~lar, there may be a change in the rate determining step with pH. It should be recognised that Km may change with pH in an independent manner to the V max as it usually involves other, or additional, ionisable groups. It is clear that at lower non-saturating substrate concentrations the activity changes with pH mayor may not reflect the changes in V max'

Enzymes

2.17

It should also be noted from the foregoing discussion that the variation of activity with pH depends on the reaction direction under consideration. The pHoPtlmum may well be different in the forward direction from that shown by the reverse reaction. This is particularly noticeable when reactions which liberate or utilise protons are considered (e.g. dehydrogenases) where there may well be greater than 2 pH units difference between the pHoptimum shown by the rates of forward and reverse reactions.

Vmax

V"

~

,

I

= ~

I

I

.........

I

\

\

J

\

, \

1

I[

~

/. 2

3

4

5

6

7 pH

8

9

\

\

\

"10

11

12

Fig. 2.6 : A generally applicable schematic diagram of the variation in the rate of an enzyme catalysed reaction (Vmax) with the pH of the solution. The centre (optimum pH) and breadth of this 'bell-shaped' curve depend upon the acid dissociation constants of the relevant groups in the enzyme. It should be noted that some enzymes have pH-activity profiles that show little similarity to this diagram. The variation of activity with pH, within a range of 2-3 units each side of the pI, is normally a reversible process. Extremes of pH will, however, cause a time- and temperature-dependent, essentially irreversible, denaturation. In alkaline solution (PH > 8), there m~y be partial destruction of cystine residues due to base catalysed ~-elimination reactions whereas, in acid solutions (PH < 4), hydrolysis of the labile peptide bonds, sometimes found next to aspartic acid residues, may occur. The importance of the knowledge concerning the variation of activity with pH cannot be over emphasised. However, a number. of other factors may mean that the optimum pH in the V max-pH diagram may not be the pH of choice in a technological process involving enzymes. These include the variation of solubility of substrate(s) and product(s), changes in the position of equilibrium for a reaction, suppression of the ionisation of a product to facilitate its partition and recovery into an organic solvent, and the reduction in susceptibility to oxidation or microbial contamination. The major such factor is the effect of pH on enzyme stability. This relationship is further complicated by the variation in the effect of the pH with both the duration of the process and the temperature or temperature-time profile. The important parameter derived from these influencp"'4is the productivity ofthe enzyme (i.e. how much substrate it is capable of converting to product). The variation of productivity with pH may be similar to that of the V max-pH relationship but changes in the substrate stream composition and contact time may also make some contribution. Generally, the variation must be determined under the industrial process conditions. It is possible to alter the pH-activity profiles of enzymes. The ionisation of the carboxylic acids involves the separation of the released groups of opposite charge.

2.18

LJ/()()/'fjnllir

Chemistry

This process is encouraged within solutions of higher polarity and rcduced by less polar solutions. Thus, reducing the dielectric constant of a aqueous solution by the addition of a co-solvent of low polarity (e.g. dioxan, ethanol), or by immobilisation, increass the pKa of carboxylic acid groups. This method is sometimes useful but not generally applicable to enzyme catalysed reactions as it may cause a drastic change on an enzyme's productivity due to denaturation. The pKa of basic groups are not similarly affected as therc is no separation of charges when basic groups ionise. However, protonated basic groups which are stabilised by neighbouring negatively charged groups will be stabilised (i.e. have lowered pKa) by solutions of lower polarity. Changes in the ionic strength (l) of the solution may also have some effect. The ionic strength is defined as half of the total sum of the concentration (c) of every ionic species (i) in the solution times the square of its charge (z) i.e. I = 05I:(c i z/). 0.2

For example, the ionic strength of a 0.1 M solution of CaCl 2 is 0.5 x (0.1 x 12) = 0.3 M.

22

+

X

At higher solution ionic strength, charge separation is encouraged with a concomitant lowering of the carboxylic acid pKas. These changes, extensive as they may be, have little effect on the overall charge on the enzyme molecule at neutral pH and are, therefore, only likely to exert a small influence on the enzyme's isoelectric point. Chemical derivatisation methods are available for converting surface charges from positive to negative and vice-versa. It is found that a single change in charge has little effect on the pH-activity profile, unless it is at the active site. However if alllysines are converted to carboxylates (e.g. by reaction with succinic anhydride) or if all the carboxylates are converted to amines (e.g. by coupling to ethylene diamine by means of a carbodiimide) the profile can be shifted about a pH unit towards higher or lower pH, respectively. The cause of these shifts is primarily the stabilisation or destabilisation of the charges at the active site during the reaction, and the effects are most noticeable at low ionic strength. Some more powerful, methods for shifting the pH-activity profile are specific to immobilsed enzymes. The ionic strength of the solution is an important parameter affecting enzyme activity. This is especially noticeable where catalysis depends on the movement of charged molecules relative to each other. Thus both the binding of charged substrates to enzymes and the movement of charged groups within the catalytic 'active' site will be influenced by the ionic composition of the medium. If the rate of the reaction depends upon the approach of charged moieties the following approximate relationship may hold, (1.20)

where k is the actual rate constant, ko is the rate constant at zero ionic strength, ZA and ZB are the electrostatic charges on the reacting species, and/is the ionic strength of the solution. If the charges are opposite then there is a decrease in the reaction rate with increasing ionic strength whereas if the charges are identical, an increase in the reaction rate will occur (e.g. the rate controlling step in the catalytic mechanism of chymotrypsin involves the approach of two positively charged groups, 57histidine+ and 145arginine+ causing a significant increase in kcat on increasing the ionic strength of the solution). Even if a more complex relationship between the rate constants and the ionic strength holds, it is clearly important to control the ionic strength of solutions in parallel with the control of pH.

Enzymes

2.19

Effect of Temperature and Pressure Enzyme action is greatly affected by temperature. 1ft he temperature is increased by lOoC the rate of most chemical reactions is doubled. IIowever, at 40°C-60°C there is loss of enzyme activity because denaturation of proteins occurs at this temperature. The rates of all reactions, including those catalysed by enzymes rise with increase in temperature in accordance with the Arrhenius equation: Where k is the kinetic rate constant for the reaction. A is the Arrhenius constant. also known as the frequency factor, ~G is the standard free energy of activation (kJ M~l) which depends on entropic and enthalpic factors, R is the gas law constant and T is the absolute temperature. Typical standard free energies of activation (15 - 70 kJ M~l) give rise to increases in rate by factors between 1.2 and 2.5 for every lOoC rise in temperature. This factor for the increase in the rate of reaction for every lOoC rise in temperature is commonly denoted by the term QIO (i.e. in this case QlO is within the range 1.2 - 2.5). All the rate constants contributing to the catalytic mechanism will vary independently, causing changes in both Kill and Vmax' It follows that, in an exothermic reaction, the reverse reaction (having a higher activation energy) increases more rapidly with temperature than the forward reaction. This, not only alters the equilibrium constant but also reduces the optimum temperature for maximum conversion as the reaction progresses. The reverse holds for endothermic reactions such as that of glucose isomerase where the ratio of fructose to glucose, at equilibrium, increases from 1.00 at 55°C to 1.17 at 80°C. In general, it would be preferable to use enzymes at high temperatures in order to make use of this increased rate of reaction plus the protection it affords against microbial contamination. Enzymes, however, are proteins and undergo essentially irreversible denaturation (i.e. conformational alteration entailing a loss of biological actIvity) at temperatures above those to which they are ordinarily exposed in thou' natural environment. These denaturing reactions have standard free energies of activation of about 200 - 300 kJ mole-1 (QlO in the range 6 - 36) which means that,

100 ~

~

..... ...,::>

80 60

S ;:j .....S 40 >': eli

S

'#. 20

,, ~ ,, ,

"

C)

eli

?''' ~'"

f l/'

~

o o

--

/

10

I'

/

V

/

"

~.

U

. .\

,,

,,

•• ••

;+-

:~o

Temperature

50

·· 60

70

(0C)

Fig. 2.7 : A schematic diagram showing the effect of the temperature on the activity of an enzyme catalysed reaction short incubation period; long incubation period. Note that the temperature at which there appears to be maximum activity varies with the incubation time.

Bioorganic Chemistry

2.20

above a critical temperature, there is a rapid rate of loss of activity (Fig. 2.7). The actual loss of activity is the product of this rate and the duration of incubation (Fig. 2.8). It may be due to covalent changes such as the deamination of asparagine residues or noncovalent changes such as the rearrangement of the protein chian. Inactivation by heat denaturation has a profound effect on the enzymes productivity (Fig. 2.9). 100

....,....., 80 ..... ...., ~ (.)

ce 60

S ;::l S ..... 40 >< ce

S

'#.. 20

0 0

10

20

30

40

50

Time (min) Fig. 2.8 : A schematic diagram showing the effect of the temperature on the stability of an enzyme catalysed reaction. The curves show the percentage activity remaining as the incuOatIOn period increases. From the top they represent equal increases in the incubation temJlerature (50°C, 55°C, 60°C, 65°C and 70°C).

..., ..... .~ ...,

>,

(.)

;::l

60°C 55°C 65°C

"0

c

;...

0..

Time Fig. 2.9 : A schematic diagram showing the effect of the temperature on the productivity of an enzyme catalysed reaction. 55°C; 60°C; 65°C. The optimum productivity is seen to vary with the process time, which may be determined by other additional factors (e.g. ,overhead costs). It is often difficult to get precise control of the temperature of an enzyme catalYRcd process and, under these circumstances, it may be seen that it is prudent to err on the low temperature side. Concentration Enzyme concentration affects the rate of a rcaction. If the substrate concentration is increased the rate of enzyme reaction also rises. Beyond a certain point, however, the

Enzymes

2.21

enzyme becomes saturated with substrate molecules. Further increase in reaction velocity occurs only if the enzyme concentration is increased. For example, during starvation the supply of the substrate glucose decreases and glycolysis is depressed. Conversely increase in glucose concentration accelerates the rate of reaction upto the point when enzyme is saturated with glucose. Inhibition Certain compounds (e.g. drugs, poisons) combine with an enzyme but do not serve as substrates (Fig. 2.10). They thus block catalysis by the enzyme and function as inhibitors. The inhibitors usually closely resemble the substrate in structure. The enzyme and the inhibitor form an enzyme-inhibitor complex which is inactive. Inhibition may be competitive or non-competitive. In competitive inhibition both inhibitor and substrate molecules compete for binding with the enzyme. If the inhibitor is in sufficiently high concentration it displaces the substrate molecules. Competitive inhibition can be reversed by increasing the concentration of the substrate. In non~ competitive inhibition the inhibitors (poisons) react with the various functional groups of the enzyme. They inhibit the normal reactions catalysed by the enzyme and result in death. Non-competitive inhibition cannot be reversed by increasing the concentration of the substrate. A

Enzyme Substrate

B Enzyme

~

Enzyme-Substrate Enz Products

... :P1oX ~b:; ~ ~ +-

complex

Inhibitor

Fig. 2.10 : (A) Enzyme action, (B) Inhibition Control of Enzyme Action The action of an enzyme can be controlled by its product (feed-back). Accumulation of the product slows down the rate of chemical reaction. There is, however, a more complicated system of control in linked systems. Many reactions in the cell take place in a series of steps, each step being controlled by a separate enzyme. For example, in the series: 81 _--=E:.:..l_~) 82 _--=E=.2_~) 83 _--=E:.:..3_~) 84

_--=E..:,.4_~)

85 _--=E..:..5_~) etc.

The substrate 81 gives rise to the product 82 through a reaction controlled by the enzyme E 1.82 now becomes the substrate of another reaction controlled by the enzyme E2, and so on.

Bioorganic Chemistry

2.22

In the synthesis of different amino acids from aspartate, lysine is formed after several steps. Aspartate -+ Aspartyl phosphate -+

I

Aspartyl -+ Lysine semialdhyde

I

It has been found that the addition of lysine stops the whole chain of reactions. Here inhibition is produced not in the last step of the series, but in the first step. Thus lysine affects the action of an enzyme which is producing an entirely different substance.

® Inctive

Control site

Control site . (Occupied)

Fig. 2.11 : Model for the action of an allosteric enzyme. This has been explained by suggesting that the enzyme has a 'control site' in addition to the 'active site' (Fig. 2.11). When the control site is occupied by a control substance (lysine, in this case), the latter distorts the shape of the enzyme. One or more reactive amino acids are removed from the active site, which is thus rendered nonfunctional. Enzymes which undergo this type of behaviour are called allosteric enzymes. They control an entire series of reactions through the end product. Isozymes Enzymes having similar biological action may be isolated from different sources. Thus many different molecular species of an enzyme have the same function. Such enzymes which exist in more than one form are called isozymes. More than a hundred enzymes are known to exist as isozymes. When the variants of an enzyme are within the same species of an organism they are called intraspecific or ontogenetic variants. When they are from different species they are called interspecific or phylogenetic variants. Multiple forms of the same enzyme may also be found in the same organism. In humans there are three different types of haemoglobin, two in the adult and one in the foetus. These are formed by the union of four polypeptide chains. The enzyme lactic dehydrogenase (LDH) is present in most animal tissues as five isozymes. It has two subunits or monomers, the H (heart) polypeptide and the M (muscle) polypeptide. Heart and muscle LDH have different amino acid compositions and different properties. Four units, in different combinations, make up the five different molecular forms of LDH. The five isozymes are :

2.23

Enzymes 1. HHHor H4 ;

2. HHHM or HaM; 3. HHMM or Hl M 2 ; 4. HMMM or HM3 ; 5.MMMMorM..

1

2

3

4

5

a~~~rn Fig. 12 : Molecular forms of LDH.

The M subunit is formed under conditions of low oxygen tension, and the H subunit under conditions of plentiful oxygen supply (aerobic metabolism). It has been found that embryos metabolize through low oxygen supply mechanisms, and in the course of development the metabolism becomes more oxygen dependent. As development proceeds the M4 and HMa forms of LDH diminish, and are replaced by the H4 and H3M forms. Isozymes may be homologous or analogous. Homologous isozymes have essentially similar molecular structure and catalystic properties. Cytochrome c from different organisms shows homology. Half the amino acids of cytochrome c of such widely separated organisms like yeasts and mammals are identical. Generally isozymes of more closely related organisms show greater similarity than isozymes of widely separated organisms. For example cytochrome c from humans and moths have 75 common residues, while those from humans and yeasts have 65 common residues. Allalogous isozymes have similar reaction but different molecular structure and catalytic properties. They have arisen from different ancestral genes. Isozymes provide a clue to the genetic relationships of organisms. The sequence of amino acids in an enzyme is related to the structure of DNA. Therefore similarity in isozymes is correlated with similarity in DNA structure. Mechanism of Enzyme Catalysis

In order for a reaction to occur, reactant molecules must contain sufficient energy to cross a potential energy barrier, the activation energy. All molecules possess varying amounts of energy depending, for example, on their recent collision history. but, generally, only a few have sufficient energy for reaction. The lower the potential energy barrier to reaction, the more reactants have sufficient energy and, hence, the faster the reaction will occur. All catalysts, including enzymes, function by forming a transition state, with the reactants, of lower free energy than would be found in the uncatalysed reaction (Fig. 2.13). Even quite modest reductions in this potential energy barrier may produce large increases in the rate of reaction (e.g. the activation energy for the uncatalysed breakdown of hydrogen peroxide to oxygen and water is 76 kJ M-l whereas, in the presence of the enzyme catalase, this is reduced to 30 kJ M-l and the rate of reaction is increased by a factor of 108 , sufficient to convert a reaction time measured in years into one measured in seconds).

Bioorganic Chemistry

2.24

~

's="'

Q> Q> Q>

Jl ,..

~

til

~

E+S

s=

.5

00.

E+P final state

Course of reaction Fig. 2.13 : A schematic diagram showing the free energy profile of the course of an enzyme catalysed reaction involving the formation of enzyme-substrate (ES) and enzyme-product (EP) complexes, i.e.. E +S

~

ES

~

EP

~

E+P

The catalysed reaction pathway goes through the transition states TScl' TS c2 and TS c3' with standard free energy of activation ~Gc*" whereas the uncatalysed reaction goes through the transition state TS u with standard free energy of activation ~Gu *. In this example the rate limiting step would be the conversion of ES into EP. Reactions involving several substrates and products, or more intermediates, are even more complicated. The Michaelis-Menten reaction scheme would give a similar profile but without the EP-complex free energy trough. The schematic profile for the uncatalysed reaction is shown as the dashed line. It should be noted that the catalytic effect only concerns the lowering of the standard free energy of ·activation from ~Gu* to ~G/ and has no effect on the overall free energy change (i.e. the difference between the initial and final states) or the related equilibrium constant: There are a number of mechanisms by which this activation energy decrease may be achieved. The most important of these involves the enzyme initially binding the substrate(s), in the correct orientation to react, close to the catalytic groups on the active enzyme complex and any other substrates. In this way the binding energy is used partially in order to reduce the contribution of the considerable activation entropy, due to the loss of the reactants' (and catalytic groups') translational and rotational entropy, towards the total activation energy. Other contributing factors are the introduction of strain into the reactants (allowing more binding energy to be available for the transition state), provision of an alternative reactive pathway and the desolvation of reacting and catalysing ionic groups. The energi

Another group include the organophosphorus compounds which undergo reaction with essential -OH groups (in serine side chains) of some enzymes. An example is diisopropylphosphofluoridate (DFP), which is a nerve poison because one of the enzymes it inactivates is acetylcholincRterase, which is important in nerve function: OCH(CHg)o)

I

E-OH + F-P

-+

I

OCH(CHg)2 enzyme

?CH(CH a)2

~

E-O-P=O

I

+ HF

OCH(CH a)2

DFP

Irreversible inhibitors are useful in the investigation of the active site of an enzyme, since the inhibitor, unlike the substrate, will remain firmly bound to one of the amino acids of the enzyme and thus act as a marker to enable it to be identified (Chapter 10). Some organophosphorus compounds are also used as insecticides.

"This page is Intentionally Left Blank"

•

CHAPTEI

MECHANISM OF ENZVME ACTION .. 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

I

I

II

Transition State Theory

1. Collision Theory Molecules can react only provided they come into contact with each other. Thus any factor which increases the rate of collisions, e.g. increased concentration of the reactants or increased temperature, will increase the rate of the reaction (according to the principles of Arrhenius and van't Hoff). Also, not all colliding molecules react. This could be partly explained by steric reasons, since not all collisions will result in the appropriate groups of molecules coming into contact, especially provided if the reactants are complex. A further and more important reason is that not all colliding molecules have between them sufficient energy to undergo a reaction.

2. Activation Energy and the Transition-State Theory Not all molecules of the same type will have the same amount of energy, taking all forms of energy into account. The energy of an individual molecule is dependant for example, on what collisions that molecule has recently been involved in. For a reaction to take place, colliding molecules must possess sufficient energy to overcome a potentialbarrier called the energy of activation. This is true even of energetically favourable reactions, i.e. those which can take place spontaneously with liberation of free energy. The fact that a reaction can, in general, take place spontaneously does not imply it will necessarily do so in all circumstances. By way of analogy, consider a ball on a hillside. The ball would tend to roll downhill; it would not spontaneously roll uphill. Nevertheless, if a stone was in its path, the ball would stay where it was. The stone would be the potential-barrier which would have to be overcome before that ball could roll down the hill. It is a similar potential-barrier which stops human beings bursting spontaneously into flame, since we burn with a great liberation of energy, if heated to a high-enough temperature. The best explanation for the requirement for activation energy in a chemical reaction is the transition-state theory which was developed by Eyring. This postulates that every chemical reaction takes place via the formation of an unstable intermediate between reactants and products. Consider the hydrolysis of an ester: (3.1)

.

3.2

Bioorganic Chemistry A

O

R'C~

+ R"OH

'OH It is accepted that the reaction mechanism involves the addition of water to the ester to form a transition-state compound possessing regions of positive and negative charge which cannot be stabilized: R'

I O=C I

/H

+ 0

R'

-.

~ I 15+/H

O=C·····O

I

,

R'

-.

~

I I

+ R"OH

O=C

'H OR" H OR" OH An unstable compound, by definition, will break down to yield a more stable one and so must have more free energy than the stable compound. A free energy profile of a reaction involving an unstable transition-state, like the one given above, is depicted in Fig. 3.1

I

transition-state

free energy

I

activation energy E"

-------------------------initial state

overall free energy change

final state course of reaction Fig. S.l : Free energy changes for an energetically favourable reaction proceeding via the formation of a transition-state.

Therefore, activation energy refers to the energy required to form the transitionstate from the reactants. The transition-state is unstable and will very quickly break down to yield the products (or back to reactants) but no products would be formed from reactants unless the transition-state gets formed. The free energy of activation thus because as a potential-barrier to the reaction occurring. An estimate of the activation energy may be found out from the reaction rate at different temperature, as will now be described. The probability that a particular molecule has energy in excess of a value E is given by e-E1HT (where R is the gas constant and T the absolute temperature). For a molecule of energy E to react it must undergo collisium with a molecule of energy at least E" - E, where E" refers to the activation energy. (To be more accurate the free energy of activation, ilG", should be used instead of E"). The probability for a colliding pair to possess sufficient energy to react is as follows :

Mechanism of Enzyme Action

3.3

-E/RT -(E'" -E)/RT

e.e

= e-E'" /RT

This has been consistent with the Arrhenius equation, which was first derived experimentally in 1889 : h

= constant

x e

-E"'/RT

(where k refers to the rate constant, which is a characteristic of the reaction at temperature T; the other constant has been equal to PZ, where P refers to a steric factor and Z the collision frequency. On taking logs, we get loge h

= loge PZ

When the rate constant

log, [

-

(:~)

= kTI at

absolute temperature T I and

= 1q.2 at

absolute temperature T 2,

~: ) = log, "'r, - log, "'r,

E '"

= R (TIT2) T _ T. loge 1

2

= [lOg, PZ -

:;, ) - [lOg, PZ -

i~1:,-1

[/q.I) -"- = 2.303 R (T TIT2) _ T. loglO [/q.I) -,'lT2 'lT2 1

2

If it is assumed that the rate of reaction is proportional to the rate constant, then we can write

(where UTI denotes the rate of reaction at temperature T I and reaction at temperature T 2)'

uT2

denotes the rate of

If the rate of reaction (or the rate constant) has been measured at more than two temperatures, E'" can be known by plotting loglok (or loglO u) against liT. The slope of the graph is - E"'/2.303 R. It is important to remember that the activation energy calculated is dependant on the assumptions made and cannot be employed to decide between possible reaction mechanisms. Acid-Base Catalysis Acids are able to catalyse reactions by temporarily donating a proton while bases might do the same by temporarily accepting a proton. It is known that a base could catalyse the hydrolysis of an ester by stabilizing the transition-state. Bases can also

3.4

Bioorganic Chemistry

increase reaction rates by enhancing the nucleophilic character of the attacking groups. Hence hydroxide ions displace halides from alkyl halides more rapidly than do neutral water molecules. In a similar way, acids facilitate the removal of leaving groups, where these are strong bases. For example, the reaction given below would normally take place extremely slowly : R3 C~O-R'

t

X"

In the presence of an acid, however, conditions have been much more favourable: R3 C-O-R' + H+

---+

R Cn-R

)'1

X-

H

.

---+

In a similar manner, in the case of ester hydrolysis, protonation of the substrate gives rise to a transition-state which avoids the unstable two-charge arrangement taking place neutral conditions, thereby increasing the rate of reaction :

......-

---"

products

In general, acid or base catalysis may be shown to be operating by measuring the rate constants of a reaction at different concentration of acid or base. Electrostatic Catalysis

A transition-state could be stabilized by electrostatic interaction between its charged groups and charged groups on a catalyst. Hence, the positive charge on a carbonium ion could be stabilized by interacting with a negatively charged carboxylate ion; similarly, the negative charge on an oxyanion could be stabilized by a positively charged metal ion. For example, the hydrolysis of glycine esters

o

0

II

II

H 2N.CH2 C.OCH3 + H 20 ? H 2N.CH 2C.OH + CHaOH could be catalysed by cupric ions, the mechanism probably has been involving the following steps :

..........~~ Cu R.N····· ·····0--. 2 \ /I HC-C-OCH 2 I a +OH2

products

MechtJnism of Enzyme Action

3.5

Covalent Catalysis As compared to acid-base and electrostatic catalysis, where the transition-state is simply modified, covalent catalysis evolves a different reaction mechanism and is sometimes known as alternative pathway catalysis. In nucleophilic catalysis, the catalyst is more nucleophilic than the normal attacking groups and so rapidly forms an intermediate which itself rapidly breaks down to yield the products. For example, according to Bender and co-workers (1957) a variety of tertiary amines catalyse the hydrolysis of esters, as given below :

O~

R~b-OR" ~ I 3

R'

+

O~ I.....~

R N-C..L.OR" 3

I R'

o

~

+

II I R'

H 0

2 R N-C + -OR" --+ 3

In contrast, electrophilic catalysts may act by withdrawing electrons from the reaction centre of an intermediate and may be called electron sinks. This type of catalysis occurs in the reactions of the coenzymes thiamine pyrophosphate and pyridoxal phosphate. Sometimes, metal ions may also be acting in this way, as in the example in electrostatic catalysis where Cu 2+ tends to facilitate the withdrawal, of electrons from the reacting carbon atom. Mechanism of Reactions Catalysed by Enzymes without Cofactors Introduction Enzymes operating without cofactors are relatively small and are having relatively straightforwards reaction mechanisms. For these reasons, such enzymes are the fIrst to be investigated in detail. Some examples are given as follows :

1. Chymotrypsin Chymotrypsin is produced by the cleavage of numerous peptide bonds in the inactive monomeric protein chymotrypsinogen, which is synthesized and secreted by mammalian pancreas; the active enzyme thus formed consists of three non-identical polypeptide chains. Chymotrypsin is able to catalyses the cleavage of peptide bonds at the carboxyl side of aromatic amino acid (phenylalanine, tyrosine or tryptophan) residues; it will also hydrolyse many amides and esters, and these -artifIcial substrates are used to investigate the enzyme in great detail. The kinetic evidence shows that the chymotrypsin-catalysed hydrolysis of an ester proceeded via the formation of an acyl-enzyme. This has been also true for the hydrolysis of amides, and one can write the reaction in general terms as follows:

E + RCO.Y -+ E.CO.R ester or amide

T

E.CO.R

YH

acyl enzyme

HP~ E + RCOOH

Enough evidence is available which shows that the substrate can bind to serine195; and histidine-57 was also implicated in the reaction mechanism. X-ray diffraction studies showed a hydrophobic binding pocket at the active site for aromatic side chains,

Bioorganic Chemistry

3.6

and revealed that aspartate~102, buried in the interior of the molecule, could be closely linked to the action of histidine-57 and serine-195, possibly setting up what has beell called a charge relay system, with aspartate-102 removing a proton from histidine-57, making it easier for the latter for removing a proton from serine-195 during the course of the reaction. Detailed lH, l3C and l5N nmr studies revealed that histidine-57 is having a relatively normal pKa value near 7, at least in the free enzyme, casting doubt on the charge relay theory. However, investigations using site-directed mutagenesis have shown that the three amino acids, if not setting up a charge relay system as such, nevertheless act in a concerted fashion, probably explaining why several enzymes are having a 'catalytic triad' of this type at the active site. The reaction mechanism for the hydrolysis of an ester or amide by chymotrypsin (or other serine protease) is given as follows. Histidine-57 behaves as a base catalyst to enable the oxygen of serine-195 cause a nucleophilic attack on the carboxyl group of the enzyme-bound substrate. An unstable tetrahedryl intermediate gets formed whose negatively charged oxygen atom may be stabilized by hydrogen bonding with the backbone -NH of glycine-193. In a similar manner, the positive charge on the imidazole ring of histidine-57 gets stabilized by electrostatic interaction with aspartate-102. The imidazole group of histidine-57 then behaves as an acid catalyst to facilitate the liberation of the first product (YH), leaving behind the acyl enzyme. ·.. ·R,

o

~ ~ 195

OH

C

~

··.. R, £,0C 0/ ~y

.1, 'y H

.....

----'"

N

) H

N 57

)~

H

~

~~

····R-C

H

-?O 'OH

OH

.....

----"

free enzyme

) H

, , ~H

····R, ,00C~

O'\'OH

,

~

acyl enzyme

#N H

The second stage of the reaction, similar to the fist, gets initiated by a nucleophilic attack, this time by water; the liberation of the product (RCOOH) is again assisted by acid catalysis. The same mechanism may take place for peptide substrates R' .NH.C(R"). CO.NH.R'" : if the substrate is again written in the form R.CO.Y, to be compatible with

Mechanism of Enzyme Action

a.7

the illustration above, R - denotes R'.NH.C(R") - (where R" is the side