UNIT 4 ENZYMES AND COENZYMES

4.1 Introduction '

4.2 Introduction to Enzymes and Coenzymes
4.3 Nomenclature and Classification of Enzymes
4.4 Specificity of Enzymes
4.5 Mechanism of Enzyme Action
4.6 Enzyme Kinetics
4.7 Factors Affecting Enzyme ~ c t i v i t y
4.8 Enzyme Inhibition
4.9 Role of Enzymes and Coenzymes in Metabolism
4. I0 lsozymes
4.1 1 Enzymes in Clinical Diagnosis
4.12 Let Us Sum Up
4.13 Glossary
4.14 Answers to Check Your Progress Exercises

4.1 INTRODUCTION
Enzymes and coenzymes are two of the most important groups of biomolecules,
without whose active participation, none of the nutrients discussed in the last three
units, can be utilized by the body. As a learner of dietitics, it is therefore essential for
you to get the knowledge of different structural and functional aspects of enzymes
and coenzymes. This unit focuses on these aspects which will enable you to
understand the metabolism of different nutrients which are to be discussed in
subsequent units of this course. Remember, this unit along with the next three units
forms the basic component of this course.

After studying this unit, you will be able to:

classify enzymes and coenzymes,
discuss their chemical nature and functions,
describe the factors on which enzyme activity depends, and
explain the diagnostic use of enzymes.

4.2 INTRODUCTION TO ENZYMES AND COENZYMES

Let us start by understanding what enzymes are. Enzymes are the proteins that
calalyze biochemical reactions. Study of these important biochemical reactions was
started many years ago, from the time of Louis Pasteur, who for the first time
demonstrated the fermentation of glucose by yeast. The catalytic agent of yeast cell
was subsequently identified and named as ferment. At the end of nineteenth century,
a cel I-free extract of yeast was found to be capable of fermenting glucose by Buchner
brothers. Considerable advances were made since then in order to properly know the
nature of these agents named as enzymes and finally their true nature was revealed by
James Summer in 1926 after the extraction and crystallization of the enzyme urease
from jack beans. At present, no less than 150 enzymes have been prepared in
crystal1ine form.

What is the chemical nature of enzymes? Let's find out. We all know that enzymes
are proteins. Do you recall the structure of proteins described in Unit 2 earlier? .
Enzymes are high molecular weight compounds made up principally of
chains of anfino acids linked together by peptide bonds as shown in Figure 4.1.

89
Cofactor Organic molecules or ions that assist many enzymes in their
reactions.
Apoenzyme The protein portion of an enzyme requiring a cofactor for its
P
reaction. P

Haloenzyme A whole enzyme, as a complete and functional molecule.

Generally, a holoenzyme consists of a polypeptide portion (an
apoenzyrne) and at least one cofactor or another coenzyme.
Enzyme Proteins that act as catalysts, speeding the rate at which
biochemical reactions proceed but not altering the direction or
nature of the reactions.
Coenqyme An organic, nonprotein molecule that binds with an apoenzyme
(a protein molecule) to form an active enzyme. Coenzymes are
often derived from vitamins.
Endoenqyme An enzyme which is not secreted or exported out of the cell,
but is kept and used by the cell which made it.
Substrate The specific molecule an enzyme acts upon.
Metabolism All of the organized chemical reactions in a cell which are
under the control of enzymes. %

Catabolic Reactions in which chemical compounds are broken down.

reactions
Anabolic Reactions in which chemical compounds are synthesized.
reactions
Enzyme The attachment of an enzyme to a solid matrix so that it cannot
immobilization escape but can still act on its substrate. .
Enzyme The disappearance of activity of an enzyme (in vivo or in vitro)
inactivation due to presence of inhibitor molecules or inhibitory conditions
(changes in pH, temperature, salt concentration etc.).
Enzyme kinetics Quantitative characteristics of enzymatic reactions. Enzymes and
Coenzvmes
Enzyme Reducing the chances that an enzyme will inactivate (in vivo or
stabilization in vitro) by changing the environmental conditions (such as
pH, temperature, concentration of salt etc.) or by attaching
organic groups to it or changing some of its amino acid
subunits.
Nutritional Biochemistry In order to follow a systematic nomenclature for all the enzymes and to classifL them,
the International Union of Biochemistry (IUB) has established a system, whereby, the
enzymes are placed into one of the six major classes as summarized in Table 4.1.
Each class is then subdivided into several classes, which are further subdivided.

The system for classification of enzymes also serves as a basis for assigning code
numbers to them. The code numbers are prefixed by EC (Enzyme Commission) and
contain four numbers separated by points, with the following meaning:
(a) the first number shows to which of the six main classes an enzyme belongs
(b) the second figure indicates the sub-class
(c) the third figure gives the sub-subclass, and
(d) the fourth figure is thc serial number of the enzyme in its sub-class.

So you can see that a number is assigned to each class, sub-class ~ndsub-subclass so
that an enzyme gets a four digit number. The fourth digit, in fact, identifies a specific
enzyme. For example, a1cohol:NAD oxidoreductase is assigned the number 1.1.1.1
because it is an oxidoreductase, the electron donor is an alcohol and the acceptor is
the coenzyme NAD. Tht~s,in naming an enzyme, the substrate is staicd first followed
by the reaction type. The trivial name of the enzyme is alcohol dehydrogenase.
Similarly, the EC number of catalase is EC 1.11.1.6. The first digit (1) indicates that
the enzyme belongs to oxidoreductase (class 1). ECl. 1 indicated subsequent digits
representing sub-classes and sub-subclasses.

Major classes of enzymes and the types of reaction catalyzed by them are
summarized in Table 4.1.

Table 4.1: Major classes of enzymes and the types of reaction catalyzed by them

Enzyme General Specific reactions catalyzed

class reaction by the member of the class
catalyzed determining the subclass
by the class
Oxido- Oxidation and 1. Dehydrogenases: Catalyze removal of two
reductases reduction of atoms of hydrogen.
(EC 1 the substrate. 2. Oxidases: Catalyze reduction of molecular
oxygen.
3. Oxygenases: Catalyze incorporation of
molecular oxygen into the substrate.
4. Oxidative deaminases: Catalyze the
oxidation of amino compounds with the
formation of ammonia.
5 . Hydroxylases: Catalyze the introduction of
hydroxyl radical into the substrate.
6. Peroxidases: Act on hydrogen peroxide.
Transferases Transfer of 1. Aminotransferases: Catalyze exchang: of
(EC2) groups amino and keto group between amino and
between two keto acids.
substrates 2. Kinases: Catalyze the transfer of pf os,)hate
radical.
3. Acyltransferases: Catalyze the tran:>feiof
acyllacetyl groups to an acceptor.
4. GIycosyltransferases:Catalyze the transfer
of glycosyl groups.
Hydrolases Hydrolysis of 1. Peptidases: Catalyze hydrolysis of peptide
(EC3) the substrate bonds.
2. Glycosidases: Catalyze hydrolysis of
glycosidic bond;
3. Esterases: Catalyze hydrolysis of carboxylic
acid esters.
92
 4. Phophatases: Catalyze hydrolysis of Enzymes and
phosphoric acid esters. Coenzvma
5 . Phosphodiesterases: Catalyze hydrolysis
of phosphodiester bonds.
6. Deaminases: Catalyze hydrolysis of amines.
7. Dearnidases: Catalyze hydrolysis of amides.
Lyases Removal of 1. Decarboxylases: Catalyze removal of
,
1
(Ec4) groups from carboxyl group from the substrate.
substrates non- 2. Aldolases: Catalyze removal of carbonyl
hydrolytically group from the substrate.
v Isomerases Isomerization 1 . Racemases: Catalyze the conversion of
i (EC5) of the substrate D-isomer to L-isomer and vice versa of a
compound.
2. Epirnerases: Catalyze the formation of an
epimer of the substrate.
3. Cis-trans isornerases: Catalyze the inter-
conversion of the cis and trans isomer of the
substrate.
4. Aldose-ketose isomerases: Catalyze the
conversion of aldose to ketose and vice versa.
5 . Mutases: Catalyze the intramolecular transfer
of a group.
Ligases Joining of two 1. Synthetases: Catalyze the formation of
(EC6) substrates at C-O, C-S, C-N bonds at the expense of
the expense of ATP.
the breakdown 2. Carboxylases: Catalyze the introduction of
of one pyro- carboxyl group with a C-C bond formation at
phosphate the expense of ATP.
bond of ATP

From the classification above, the different classes of enzymes identified include:
oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. Let's get to
know more about them and their nomenclature.

Oxidoreductases

To this group (EC 1) beIongs all enzymes catalyzing oxidation-reduction reactions.

Common names include dehydrogenases, oxidazes, reductases and catalases. The
substrate (AH2) that is oxidized is regarded as a hydrogen donor (AH2 + B = A +
BH?). The recommended name is dehydrogenase but, as an alternative, the term
reductase is used. The name ,oxidase is restricted to enzymes which exclusively use
02 as the hydrogen acceptor. The second figure in the code number of
oxidoreductases indicates the group in the hydrogen donor which undergoes
oxidation e.g. CH-OH, CHO, CH-CH, CHNH2, NAD(P)H. Therefore, EC 1.1
indicated acting on the CH-OH group of donors. EC 1.2 acting on the aldehyde or
0x0 group of donors etc. The third figure indicates the type of acceptor involved:
1 denotes NAD(P), 2 a cytochrome, 3 02, 4 a disulphide, 5 a quinine etc. So, EC
I . 1.1states with NAD' or NADP' as acceptor, EC 1.1.2 with cytochrome as acceptor
and so on, the list goes on.

These are the enzymes (code EC 2) which catalyze the transfer of a group, e.g. a
methyl or glycosyl group, from one compound to another. In many cases, the donor is
a cofactor (coenzyme) carrying the group to be transferred. Common names include
acetyltransferase, methylase, protein kinase and polymerase. The second figure
(EC 2.1) in the code number of transferases indicates the group transfei-red: a carbon
group (2. l), a carbonyl group (aldehyde or ketone) (2.2), a glycdsyl group (2.3) and
so on. The third figure informs of the group transferred: e.g. subclass 2.1 is
Nutritional Biochemistry subdivided into methyltransferases (2.1. I), hydroxymethyl and formyltransferases
(2.1.2) and so on.

Hydrolyases

These enzymes (code EC 3) catalyze the hydrolytic cleavage of C-O, C-N, C-C and
some other bonds, including phosphoric anhydride bonds. Their trivial names are
formed by adding the suffix ... 'ase ' to the substrate which they hydrolyze. Examples
include protease, nuclease, phosphatase. A number of hydrolases acting on ester,
glycosyl, peptide, amide or other bonds are known to catalyze not only the hydrolytic
removal of a particular group from their substrates, but also the transfer of this group
to a suitable acceptor molecule. Yet, they are not grouped as transferases, because the
transfer of a specific group to water as the acceptor molecule is considered to be their
main physiological function. The second figure in the code number of hydrolases
indicates the nature of the bond hydrolyzed, e.g. esterases (3.1), glycosidases (3.2) -
and so on. The third figure generally specifies the nature of the substrate, e.g.
carboxylic esters (3.1. I), thiol esters (3.1.2), phosphoric monoesters (3.1.3), 0-
glycosides (3.2.1), N-glycosides (3.2.2) and so on.

These enzymes (code EC 4) cleave C-C, C-O, C-N and other bonds by elimination,
forming double bonds or conversely adding groups to double bonds. Common names
include decarboxylase, aldolase, dehydratase (if water is eliminated) or hydro-lyase
(if the reverse reaction is more important or the only one which can be
demonstrated). Spthase, but not synthetase, may be used as in tryptophan synthase
or cystathionine P-synthase. The second figure in the EC number indicates the bond
being cleaved for e.g. C-C-lyases (4. I), C-O-lyases (4.2) and so 'on. The third figure
informs about the group that is eliminated e.g. COz (4.1.l) or H20 (4.2.1).

Isomerases

These enzymes (code EC 5) catalyze geometric or structural changes within a

molecule. According to the type of isomerism, they may be called racemases,
epimerases, cis-trans isomerases (5.2), isomerases, tautomerases, mutases or cyclo-
isomerases. EC 5.1 are racemases or epimerases, EC 5.1.1 indicates acting on amino
acids and derivatives and so on.

Ligases (synthetases)

These enzymes (code EC 6) catalyze the linkage of two molecules coupled with the
hydrolytic breakdown of a pyrophosphate bond in ATP or an analogous compound.
The bonds formed are often high energy bonds. The second figure in the code
number indicates the bond formed, e.g. C-O (6.1), C-S (6.2) etc. Examples include
. peptide synthase, aminoacyl-tRNA synthetase, DNA ligase and RNA ligase.

We hope the information given above may have helped you in understanding the
classification of enzymes. The system of the nomenclature and the classification of
enzymes are based exclusively on the reaction that is catalyzed and does not consider
their origin or multiplicity. Enzymes catalyzing the same reaction, but isolated from
different species will have varying amino acid sequences so that they may be
distinguished by electrophoretic methods. ,They may have different sizes and net
negative charges and may even differ in their,catalytic behavior.

With this basic knowledge about enzymes, we shall go on to leam about the
@
specificity of enzypes.
4.4 SPECIFICITY OF ENZYMES
One of the major characteristics of enzymes is that they are highly specific. Enzymes,
the organic catalysts, differ from the inorganic catalysts in their extraordinary
specificity. Each of the enzymes that have been isolated and studied is found to

Reaction specificity

Optical specificity

We shall look at each of these specificities one by one.

Reaction specijicity

i) Some enzymes catalyze only one reaction acting on a specific substrate.

Example: urease and catalase acts only on urea and hydrogen peroxide,
respectively. This is also called absolute specificity.

ii) Many enzymes can catalyze same type of reactions (phosphate transfer,
oxidation-reduction, hydrolysis etc.) in several structurally-related compounds.
Example: carboxypeptidase acts on protein chains and removes one amino acid at
a time from the C-terminal, irrespective of the nature of zimino acid.

iii) A substrate caa undergo many reactions but in a specific reaction, an enzyme will
catitlyze only one of these reactions. Example: citrate synthase converts
oxaloacetate to citrate in the presence of acetyl-CoA. But, in absence of acetyl-
CoA, oxaloacetate is acted upon by a different enzyme malate dehydrogenase
:

wit11 the formation of malate.

Bond specificity

i) Some enzymes act on a particular bond (glycosidic, peptide, ester etc.).

Examples: pepsin, trypsin, chymotrypsin etc. are all only acting on peptide
bonds, though the peptide bonds on which they will attack depend on the amino
acids that have formed the bonds.

Group speciJicity

i) Some enzymes prefer a specific functional group to be present\%on the substrate

molecules. ~ x a m ~ lalcohol
e: dehydrogenase acts on alcohols having -OH group.

ii) Some proteases act on the peptide bonds depending on the amino acids involved
in the formation of the bonds. Example: chymotrypsin hydrolyzes only the

Optical speczficity

i) Some enzymes exhibit absolute optical specificity for at least a portion of the
substrate molecule. For example, the enzyme maltase catalyzes the hydrolysis of
a-glycosidic bond between two glucose molecules but not the P-glycosidic bond.
Check Your Progress Exercise 1
1 ) Define the following terms:

a) Enzymes
............................................................................................................................
............................................................................................................................
b) Holoenzyme
.............................................................................................................................
............................................................................................................................
c) Metabolism
............................................................................................................................
............................................................................................................................
2) Differentiate between endoenzymes and exoenzymes, giving suitable
examples.
............................................................................................................................
............................................................................................................................
............................................................................................................................
3) How are e4zymes classified? Explain giving an appropriate example.
............................................................................................................................
............................................................................................................................
............................................................................................................................
4) How are enzymes different from inorganic catalysts?
..............................................................................................................................
............................................................................................................................
............................................................................................................................
5) What do you understand by enzyme specificity? List the four different types
of enzyme specificities.
............................................................................................................................
............................................................................................................................
............................................................................................................................
6) Match the following:
A B
a) Oxidoreductases i) Kinases, Glycosyltransferases
b) Isomerases ii) Synthetases, Carboxylases
C) Transferases iii) Peptidases, Phosphatases
d) Lyases iv) Racemases, Mutases, Isomerases
e) Hydrolases v) Dehydmgenases, Oxidases, Peroxidases
f) Ligases vi) Aldolases, Decarboxylases
 So far we have leant about enzymes, their chemical nature, classification and some Enzymes and
characteristics. Next, let us learn about the mechanism of enzyme action. Coennmes

'
4.5 MECHANISM OF ENZYME ACTION
L. Micl~aelisand M L. Menten developed a general theory of enzyme action in 1913.
According to Michaelis and Menten, the enzyme (E) first binds the substrate (S) to
form a transient enzyme-substrate complex (ES). This complex then dissociates into
the product (P) and the unaltered enzyme (E).

E + S ----b ES E+P
(Transient
complex)

Let us understaiid how this ES complex forms.

The action of all enzyme is initiated when the reactants i.e. substrates bind at the
catalytic sites or active sites on the enzyme molecule. The catalytic site of the
enzylne molecule possesses a complex three-dimensional form and provides a cleft,
which binds the substrate as shown herewith.

A change in the tertiary or quaternary structure of the enzyme may alter the three
dimensional shape of the catalytic site and thus reducing its binding and catalytic
activ~ties.The ES complex is formed mainly liynon-covalent bonds between specific
groups of the substrate molecules and the specific amino acid side chains present at
the catalytic site of the enzyme. Different models for enzyme-substrate complex
formation exist. Let us look at these models next.

Modelsfor enzyme-substrate (ES) complexformation

There are two popular models to explain the enzyme-substrate interaction. These are:

Fischer's template or lock and key model, and

Koshland's induced fit model

The two models are described next.

Fisc her 's template or lock and key model

According to this model, the catalytic site of the enzyme has a proper conformation
compatible to a specific substrate even in the absence of the substrate molecule, as
shown in Figure 4.3. The catalytic site binds the substrate and catalyzes the reaction
without any change in its own three dimensional conformation. It has become
possible to explain the specificity of many enzymes for only one of the stereoisomers
of the substrate by this model. This model, however, failed to explain the change in
enzylne activity in presence of allosteric modulators (low molecular weight
regulatory substances that bind at a specific site on the enzyme molecule other than
the catalytic site and thereby enhance or inhibit the enzyme activity) or the action of
the noncompetitive inhibitors, about which we will learn later in section 4.8 in

97
Nutritional Biochemistry
 After understanding tlie mechanism of enzyme action, we move on to enzyme ]Enzymes and
kinetics. What is enzyme kinetics? Let's find out. Coenzvmes

4.6 ENZYME KINETICS

The study of the rate at which an enzyme works is called enzyme kinetics.

You have read in the last section that L. Michaelis and k L. Menten developed a
general theory of enzyme action and kinetics in 1913. According to this theory, we
learnt that in an enzyme substrate reaction, the ezzyme (E)first combines with the
substrate (S) with the formation of an enzyme-substrate (ES) complex, which
subsequently breaks down into the product (P) and the enzyme (E) is recovered.
Thus, tlie enzyme catalyzed reaction proceeds in the following two steps:

KI K3
(a) E+S -ES (b) ES .- E+P
, '
K2 I<4
The reactions are assumed to be reversible and K1, K2, K3 and Kq are the rate
constants of each reaction.

Let us examine enzyme kinetics as a function of the concentration of substrate

available to the enzyme. The enzyme substrate interaction may be studied with the
help of Figure 4.5. In this graph, substrate concentration and velocity of reaction of
the enzyme catalysis has been shown on X and Y axis, respectively. When the
velocity of a reaction is plotted against different substrate concentrations, as iv the
present graph, a hyperbolic curve is obtained.

Substrate Concentration

Figure 4.5: Effect of substrate concentration on thereastion ueloc@y

of an enzyme catalyzed reaction

M, N and 0 are three points on the curve representing three stages of enzyme
catalyzed reaction. While M represents that stage of the reaction when the substrate
concentratio11 is very low and the rate of reaction is directly proportional to the
substrate concentration, 0 represents the stage when the reaction velocity reaches its
maximum due to the gradual increase in substrate concentration resulting in a
saturation of the active site df the enzyme. In between these two extremities lies the
third point N which represents the reaction velocity of the enzyme catalyzed reaction
, that is half of the maximum velocity.

99
Nutritional Biochemistry The mathematical relationship between the initial velocity of an enzyme catalyzed
reaction, the concentration of the substrate and certain characteristics of the enzyme
are expressed by the Michaelis-Menten equation, which was derived on the basis of
Michaelis-Menten theory and assumptions of G. E. Briggs and J. B. S. Haldane and
was proposed in 1925.

The Michaelis-Menten equation is:

Vm, [Sl
v=
K m + [SI
where, v is the velocity of the reaction at any stage,
V, is the maximum velocity,
[S] is the substrate concentration and
K,,, is the Michaelis-Menten constant, usually expressed in moles per litre.

K,,, represents the substrate concentration at which the velocity of the reaction is half
of V,. K,,, is (roughly) an inverse measure of the affinity or strength of binding
between the enzyme and its substrate. The lower the K,,,, the greater the affinity
(so, lower is the concentration of substrate needed to achieve a given rate).

This equation is widely used to describe the most enzyme catalyzed reactions. Now,
we can verify the situations of the three points M, N and 0 in Figure 4.5 with the
help of this equation.

i) Point M: At this stage of enzyme-substrate reaction, the substrate concentration is

much less than K,,, and its value does not changes significantly with the increase in
substrate concentration. Thus, [6] can be ignored in the denominator of the
Michaelis-Menten equation. So it becomes,

vm [SI
v=
K,,,

As V, and K,,, are both constants, ,V / K,,, ratio may be replaced by a new
constant, K. Thus, we have
v = K x [S]
So, reaction velocity (v) of the enzyme-substrate reaction at this stage is directly
proportional to substrate concentration [S].

ii) Point N: At this point, substrate concentration [S] is equal to Michaelis-Menten

constant, K,,,. Thus the Michaelis-Mefiten equation becomes:

V"=
El+ [SI
v, [SI
v=
2 [SI

v,,
or v=
2
This clearly established that K,,, is the substrate concentration at which the reaction
velocity of an enzyme catalyzed reaction is half of maximum velocity.
Check Your Progress Exercise 2

1) Discuss the general theory of enzyme action.

............................................................................................................................
............................................................................................................................
............................................................................................................................
2) What is meant by lock and key model? What are its drawbacks?
.............................................................................................................................
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............................................................................................................................
3) Give the four different ways by which the enzyme activity can be expressed.
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4) Write down Michaelis - Menten equation.
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5) Give the relationship between substrate concentration and reaction velocity.
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6) How can affinity of an enzyme for a substrate be judged?
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7) Name one model of enzyme-substrate interaction which can explain non-
competitive inhibition.
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]Enzymesand
Coenzvmes
 15nzymes and
Activators Coenzvmes

Activity of many enzymes is influenced by certain ions called as activators. Large

number of enzymes such as hexokinase that require ATF are also in need of divalent
. cations like M ~ ' + or ~ n " . Many enzymes such as ATPase require monovalent
cations like Na+, K' or N&' for maximum catalytic activity. Amylase requires C1-. -
Generally, these ions interact with the substrates so that the substrates can bind with
the catalytic sites of the enzyme properly. Thus, in absence of the activators, the
enzymes become inactive or sluggish.

Oxidation

Some enzymes which have the sulfhydryl (-SH) group in the catalytic site are very
sensitive to oxidation. Due to oxidation of the -SH group by aerial oxygen or
oxidizing agents, a disulfide linkage (-S-S-) forms with the subsequent loss of
enzyme activity. The enzyme activity can be restored by the reduction of the enzyme
by some reducing agent such as cysteine or glutathione.

Having studied about the factors influencing the enzyme activity, we move on to
factors which. inhibit the enzyme activity in the next section.

4.8 ENZYME INHIBITION

Enzymes are often inhibited by the presence of suitable inhibitors. Much of current
drug therapy is based on this. Basically, there are three major classes of enzyme
inhibition. These are:

Competitive inhibition, when the substrate and inhibitor compete for binding to
the same active site
Noncompetitive inhibition, when the inhibitor binds somewhere else on the
enzyme molecule reducing its efficiency, and
IJncompetitive inhibition

Let us learn about each of these classes of inhibition.

Competitive inhibition

This type of inhibition takes place when a compound having a strong structural
resemblance to the substrate competes with it for the catalytic site of the enzyme.
Once the compound binds, the enzyme cannot convert the inhibitor to products.
Increasing substrate concentration, however, is capable of displacing the inhibitor.
Thus this type of inhibition is reversible in nature. A good example of this is the
reaction catalyzed by succinate dehydrogenase in the citric acid cycle. You will learn
about the citric acid cycle in Unit 6, later in this Course. In this reaction, succinate is
converted to fumarate with the aid of this enzyme. Now, the compound malonate is
structurally similar to succinate and if present, it will compete with succinate for the
catalytic site of succinate dehydrogenase and reduce the product formation. Thus,
malonate is a competitive inhibitor for this particular reaction. The structure of
succinate and malonate is given in Figure 4.1 1.

COOH COOH
I I
CH2 CH2
I I
C"2 COOH
I
COOH
Succinate Malonate
Figure 4.11: Structure of succinate and malonate

105
 In this type of inhibition, the K,,, of the enzyme for the substrate shows an apparent
increase in the presence of the inhibitor as can be seen in Figure 4.12. This means
that by increasing the substrate concentration, enzyme inhibition can be overcome.
However, the V, remains unaltered.

Figure 4.12: Lineweaver-Burk plot for an enzyme-substrate

reaction in competitive inhibition

Next, let us learn about the noncompetitive inhibition.

Noncompetitive inhibition

In this type of inhibition, the inhibitor binds at a site on the enzyme other than
catalytic site. As there is no competition between the substrate and the inhibitor, the
inhibition cannot be reversed in this case by increasing the substrate concentration. It
appears that as if inhibitor is removing the enzyme, thus causing a decrease in V, as
can be seen in Figure 4.13. No change of K,, however, occurs.

Figure 4.13: Lineweaver-Burk plot for an enzyme-substrate

reaction in noncompetitive inhibition

A noncompetitive inhibitor can combine with either the free enzyme or the enzyme-
substrate complex, interfering both. The most common type of noncompetitive
inhibition is affected by the substances that combine with some functional group of
the enzyme (outside the catalytic site) that is essential for maintaining the
conformation of the enzyme molecule required for its activity. For example, enzymes
possessing the essential -SH group are sometimes inhibited by metals like mercury
or copper.

Finally, let is get to know about the third type of enzyme inhibition i.e. uncompetitive
inhibition.

106
 Uncompetitive inhibition Enzymes and
Coenzvmes
In this type of inhibition, the inhibitor only binds with the enzyme-substrate complex
making it inactive. As a result, the product formation becomes difficult. In
uncompetitive inhibition, both K, and V , ,changes as can be seen in Figure 4.14.
The former increases while the latter decreases. This kind of inhibition is rare in one
substrate reactions but common in two substrate reactions.

Figure 4.14: Lineweaver-Burk plot for an enzyme-substrate

reaction in uncompetitive inhibition

So far we studied about the enzyme mechanism and the factors which influence and
inhibit its activity. Our study of enzymes shaIl be incomplete, without the
understanding of the role of enzymes and coenzymes in metabolism. The next section
focuses on this aspect. But before moving on to the next section, let us recapitulate
what we learnt till now.

Check Your Progress Exercise 3

1 ) Name the environmentale factors on which enzyme activity depends.
Explain any two of these.
............................................................................................................................
3

............................................................................................................................
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3) Explain the following terms giving suitable examples.

a) Competitive inhibition
............................................................................................................................
............................................................................................................................
b) Uncompetitive inhibition
............................................................................................................................
............................................................................................................................
c) Non-competitive inhibition
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1 Nutritional Biochemistry 4) How does the K, of an enzyme catalyzed reaction change in the presence of
a competitive inhibitor?
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............................................................................................................................
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4.9 ROLE OF ENZYMES AND COENZYMES IN

METABOLISM
You have already learnt that many enzymes require a non-protein part for their
optimal activity, which may be a coenzyme or a metal ion. This has also been stated
earlier that many water soluble vitamins are acting as coenzymes in their native or
derived forms. So, it is important to learn how the coepzymes are assisting different
enzymes in catalyzing biochemical reactions. In this sub-section, you will learn
about:
the chemical nature of different coenzymes, and
the general role of co-enzymes in assisting different enzymes during metabolism.

As coenzymes participate in a variety of functions, they can be classified broadly into

two groups:

i) Hydrogen transferring coenzymes, and

ii) Group transferring coenzymes

Let's learn about these two groups of enzymes.

I) Hydrogen Trans@rring Coenzymes

This group consists of three important coenzymes all of which assist different
enzymes in oxidation-reduction reactions. These are:

a) Nicotinamide nucleotides
b) Flavin nucleotides
c) Lipoic acid

A brief review of these coenzymes follows.

(a) Nicotinamide nucleotides

These coenzymes are derived from the vitamin, niacin. You may recall reading in
Unit 3 that they are of two types, nicotinamide adenine dinucleotide (NAD') and
nicotinamide adenine dinucleotide phosphate (NADP'). Collectively, they are called
as pyr~dinenucleotides. Have a look at Figure 4.15. In NAD', the pyridine ring is
attached to a ribose molecule through glycosidic bond and the phosphate provides a
link between adenosine and nicotinamide riboside. In NADP', an aqditional
phosphate group is present in carbon atom 2 of the ribose molecule of adenosine
component.
Enzymes and
Coenzvmes
 Pyridoxal phoshphate Enzymes and
Coenzvmes
Pyridoxal phosphate is derived from pyridoxine (vitamin B6) and is involved in
amino acid metabolism. The other two compounds, pyridoxal and pyridoxamine,
about which you learnt in the last unit, having the properties of vitamin B6 also occur
as phosphate derivatives. Enzymes that are dependent on B6 phosphate coenzymes
catalyze a variety of reactions such as transamination (transfer of amino group from
an amino acid to a keto acid), decarboxylation (removal of carboxyl group) and
racemization (transformation of one isomer to another). The structure of pyridoxal
phosphate is shown in Figure 4.20.

Pyridoxal Phosphate (PyP; Vitamin B),

Figure 4.20: Structure of pyridoxal phosphate (Pyp; Vitamin B6)

Enzyme glutamate oxaloacetate transaminase catalyzes the reaction between

glutamate and oxaloacetate with the formation of a-ketoglutarate and aspartate due to
transfer of one amino group from glutamate to oxaloacetate. Pyridoxal phosphate
acts as the carrier of the amino group. Whenever it accepts the amino group,
it transforms to pyridoxamine phosphate, and after the release of the amino group,
it again becomes pyridoxal phosphate. Aspartate is decarboxylated by aspartate
decarboxylase taking the help of pyridoxal phosphate.

L-alanine transforms to D-alanine by alanine racemase which also requires pyridoxal

phosphate as the co-enzyme.

CoenzymeA

Coenzyme A is derived from the vitamin pantothenic acid. This is abbreviated as

CoA. This can be divided into two components, adenosine 3,5-diphosphate and
pantotheine, which is formed by the combination of pantothenic acid and
mercaptoethylamine. Refer Figure 4.21 to understand its structures well. It gives rise
to acyl-CoA derivatives that are mainly formed in ATP dependent synthetase
reactions. These are highly reactive and participate in various types of reactions. For
example, oxaloacetate is converted to citrate in presence of citrate synthetase by
accepting acyl group of acyl-CoA. During carbohydrate metabolism, the pyruvate is
converted by the pyruvate dehydrogenase complex to acetyl-CoA by the active
participation of coenzyme A. Also, in the oxidation of fatty acids, CoA has an
important role to play as you will find out in Unit 7.
Coenzyme A (CoA)
p - Alanine Pantothenate

1 - II I
2-Mercaptoethylamine Pantothenate ADP with ?-Phosphate Gn)up

Figure 4.21: Structure of coenzyme A

113
I
Nutritional Biochemistry So far we have studied about the different enzymes and coenzyme involved in
metabolism. Interestingly, it is known that enzymes catalyzing essentially the same
reaction may differ in various ways. These are called isozymes. The next section
focuses on isozymes.

4.10 ISOZYMES
Sometimes an enzyme present in the same organism is found to have different
molecular forms but catalyzing the same reaction. These are called isozymes. The
1964 Committee recommended that "multiple enzyme forms" in a single species
should be known as isozymes. Among many enzymes known to have isozymes, the
most studied is lactate dehydrogenase (LDffl, which is an important enzyme of
carbohydrate metabolism. This enzyme exists in five possible forms in most
vertebrates. In fact two basically different types of LDH occur predominantly in the
heart and muscle. The former consists of four identical monomers (H). The latter
(muscle LDH) also forms due to the combination of four identical monomers (M) but
having different amino acid composition, in comparison to the former. Different
combinations of H and M monomers yield three additional hybrid enzymes
possessing four monomers each. The possible combinations of H and M monomers
therefore, produce five isozymes of LDH. These are:

Ii4. H3M, H2M2, HM3 zmd M'l

Isozymes differ from each other not only in the amino acid composition, they also
have different electrophoretic property, thermolability, immunological properties and
kinetic properties such as substrate affinities i.e. different K,,,
values.

Estimation of isozymes of some enzymes can be used for the clinical diagnosis of
different diseases. Let us learn about this aspect next.

4.1 1 ENZYMES IN CLINICAL DIAGNOSIS

The rationale for measuring plasma or serum enzyme levels is based on the premise
that these levels reflect changes that have occurred in a specific tissue or organ.
Enzymes present in the blood are of two types - one type such as thrombin
(associated with blood coagulation) has a functional role and is present in high
concentration, the other type has no functional role in the blood and is present in very
small amount. The latter types of enzymes mainly originate from different tissues or
organs. An insult in the form of any disease may cause changes in cell membrane
permeability or increased cell death, resulting in the release of intracellular enzymes
into the blood: As a result, the concentration of these enzymes increases in the blood.
In the diagnosis of specific organ involvement in a disease process, it would be ideal
if enzymes unique to each organ could be identified. But, this seldom occurs as the
metabolism of different organs is not unique. Alcohol dehydrogenase and acid
phosphatase are two such enzymes, the levels of which in the blood can be used as
specific tools for the diseases of liver and prostrate, rsspectively. However, the ratio
of enzymes varies from organ to organ. This fact, combined with a study of the
kinetics of appearance and disappearance of particular enzymes from the blood,
enables one to identify the involvement of a specific organ in a disease. Both
glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase
(GPT) are present in the liver but the concentration of GPT is more than GOT. In
liver dysfunction, both these enzymes are leaked out from liver and as a result, their
level in the blood increases but the rise is more in case of GPT than GOT making it a
reliable marker for liver diseases.

Following a heart attack, many enzymes such as GOT, creatinephosphokinase, LDH

etc. are released from the myocardium but their time of release differs, enabling one
to establish when the attack occurred and whether the treatment is effective or not.
116
 Enzyme Normal value Concentration increases in conditions
in the serum
Acid 1-5 initsldl Metastatic carcinoma in the prostrate.
phosphatase
Alkaline 5- 13 unitsldl Rickets, hyperparathyroidism, obstructive
phosphatase jaundice, kidney disease, metastatic
carcinoma, osteoblastic sarcoma.
Isozymes of alkaline phosphatase can
distinguish liver lesions from bone lesions
in cases of metastatic carcinoma.
Amylase 0.8-32 IU& High intestinal obstruction, acute pancreatitis.
Acetyl 3-5 IUIml Nephrotic syndrome.
cholinesterase
Creatine 55 7 5 units& Muscular dystrophy, myocardial infarction.
phosphokinase (Male)
6-50 unitsll
( F e m a l e )
Glutamate- 5-40 unitsldl Myocardial infarction, slightly elevated in
oxaloacetate liver diseases.
transam inase
(SGOT)
Glutamate- 5-35 unitsldl Acute liver diseases, slightly elevated in
pyruvate cardiac necrosis.
transaminase

Lipase 0.2-1.5 unitsldl Acute pancreatitis, pancreatic carcinoma.

Glucose-6- 3.5-7.5 unitslml Haemolytic anaemia often associated with
phosphate administration of anti-malarial or
dehydrogenase sulphonamide drugs and after eating fava
(in RBC) bean.
Lactate 90-200 IU& Myocardial infarction, acute hepatitis, renal
del~ydrogenase tubular necrosis.

Check Your Progress Exercise 4

1 ) What are coenzymes? HOW are the coenzymes grouped?
............................................................................................................................
...

....... ...................................................................................................................
L

............................................................................................................................
2) Name the type of reactions in w..ich the following co-enzymes a,, .~volved.

a) NAD' and NADP' ............................................................................

b) FAD and FMN ............................................................................
c) Lipoic acid ............................................................................
d) Biotin ............................................................................
e) TDP ............................................................................
f) Tetrahydrofolate ............................................................................
3) Match the following:
A B
a) Niacin i) TDP
b) Riboflavin ii) NAD+ and NADP'
c) Thiamine iii) FAD and FMN
d) Biotin iv) Pyruvate carboxylase

4) What is the main role of tetrahydrofolate in different biochemical reactions?

............................................................................................................................
5) What are isozymes? How many isozymes of LDH are known?
............................................................................................................................
............................................................................................................................
............................................................................................................................
6) Name any two enzymes which are determined during the clinical diagnosis
of the following diseases:

a) Heart attack ............................................................................

b) Liver diseases ...........................................................................
c) Acute pancreatitis ............................................................................
 Further, in this unit we got the idea how different coenzymes are taking part in
different enzyme-catalyzed reactions with suitable examples. This will also greatly
help us in learning different metabolic reactions in the subsequent units of this
course. lmportance of serum enzymes and isozymes in the diagnosis of different
diseases is well known. This has been discussed comprehensively. Thus this unit has
covered all important aspects of enzymology but emphasis has been given only on
the topics relevant to our need as nutritional biochemists.

4.13 GLOSSARY

: the site on the surface of an enzyme to which substrate or

substrates bind.

Conformation : specific arrangement of the molecule.

Denaturation : change from original structures.
Electrophoretic : ability to move in an electric field.
: liberation of haemoglobin from erythrocyte causing

International : the amount of enzyme that catalyses the transformation of

1 p o l of substrate into product in 1 minute.
: an enzyme having different molecular forms but catalyzing
the same reaction.
: the amount of enzyme that transforms 1 mol of substrate
into product in one second.
Metabolism : synthesis or breakdown of bio-molecules.
: cancer that can be transferred from one part of the body to
other unrelated parts.
: defects in the muscle due to faulty nutrition.

: necrosis of myocardium due to interruption of blood

: the middle layer of the heart wall.

: cell death.
: kidney disease due to degeneration of renal tubule.

Osteoblastic : a kind of tumor.

sarcoma
Pancreatitis : inflammation of the pancreas.
Prosthetic group : a non-protein part of the enzyme which remains tightly
bound to the protein part.
: a condition in children due to vitamin D deficiency.
Thermolability : temperature sensitivity.
: less important.
' Turnover number : the number of molecules of substrate transformed per
catalytic site of the enzyme per minute,
Nutritional Biochemistry
4.14 ANSWERS TO CHECK YOUR PROGRESS
EXERCISES
Check Your Progress Exercise 1

1) a) Enzymes are the proteins that act as catalysts, speeding the rate at which ,
biochemical reactions proceed but not altering the direction or nature of the
reactions.
b) A holoenzyme is whole enzyme, as a complete and functional molecule.
Generally, a holoenzyme consists of a polypeptide portion (an apoenzyme)
and at least one cofactor or other coenzyme.
c) Metzbolism is all of the organized chemical reactions in a cell which are
under the control of enzymes.
2) Enzymes which act inside the cell are called as endoenzymes and those which are
liberated outside the cell are called as exoenzymes. While enzymes like those
involved in the production of energy belong to the first category, digestive
enzymes belong to the second category.
3) The enzymes are classified by assigning code numbers to them. The code
numbers are prefixed by EC (Efizyme Commission) and contain four numbers
separated by points, with the following meaning:
a) the first number (EC1) shows to which of the six main classes an enzyme
belongs
b) the second figure (EC 1.1) indicates the sub-class
c) the third figure (EC 1.1.1) gives the sub-subclass, :-
d) the fourth figure (EC 1 .l. 1.1) is the serial number of the enzyme in its sub-
class.
For example, alcohol : NAD' oxidoreductase is assigned the number 1.1.1.1
because it is an oxidoreductase, the electron donor is an alcohol and the acceptor
is the coenzyme NAD'. Thus, in naming an enzyme the substrate is stated first
followed by the reaction type.

4) Enzymes, the organic catalysts, differ from the inorganic catalysts in their
extraordinary specificity.
5) The enzymes are specific in action that is, they catalyze a particular reaction or a
particular type of chemical bond or functional group. This is referred to as
enzyme specificity. The 4 different types of specificities are reaction specificity,
bond specificity, group specificity and optical specificity.
6) a) - v)
b) - iv)
c) - i)
d) - vi)
e) - iii)
f) - ii)

Check Your Progress Exercise 2

1) The general theory of enzyme action given by Michaelis and Menten, states that
the enzyme (E) first binds the substrate (S) to form a transient enzyme-substrate
complex (ES). This complex then dissociates into the product (P) and the
unaltered enzyme (E).
E+S -----b ES -----+ E+P
2) According to lock and key model, the catalytic site of the enzyme has a proper
conformation compatible to a specific substrate even in the absence of the
substrate molecule. The catalytic site binds the substrate and catalyzes the
reaction without any change in its own three dimensional conformation. This
model, however, failed to explain the change in enzyme activity in presence of
allosteric modulators or the action of the noncompetitive inhibitors.
 Enzyma and
3) The enzyme activity can be expressed as 'katal', which is the amount of an Coenzvmes
e~lzymethat transforms 1 mol of substrate into product in one second.

Other than katal, the activity of an enzyme may be expressed in different other

Molar activity of an enzyme, expressed as katlmol of the enzyme.

Turrnover number of the enzyme.

4) The Michaelis-Menten equation is:

vm, [SI
v=
Km + [Sl
where, v is the velocity of the reaction at any stage, V , is the maximum
velocity, [S] is the substrate concentration and K, is the Michaelis-Menten
constant, usualIy expressed in moles per litre.
5) The relationship between substrate concentration and reaction velocity is best
explained through the hyperbolic curvei'when substrate concentration is low,
rate of reaction is directly proportional to the substrate concentration. A gradual
increase in the substrate concentration results in saturation of active enzyme site

v=
- Km

Here, V, and K,,, are both constants, V, 1 K, ratio may be replaced by a new
constant, K. Thus, we have:

So: reaction velocity (v) of the enzyme-substrate reaction at this stage is directly
proportional to subs%ateconcentration [S].
6) Affinity of an enzyme for a substrate can be judged by K,. The enzyme affinity
inversely varies with the K,.
7) Koshland's induced fit model can explain noncompetitive inhibition.

Check Your Progress Exercise 3

1) Concentration of enzyme, concentration of substrate temperature, pH, activators,

, oxidation are the environmental factors on which enzyme activity depends. A
brief explanation of two factors follows:
a) Concentration of enzyme: Increase in enzyme concentration will increase the
rate of an enzyme catalyzed reaction. This is.because bf the availability of
additional catalytic sites at the beginning of the reaction to which the
substrates can bind.

b) Concentration of substrate: At low concentration, the rate of reaction also

remains low. This is because at this stage all the catalytic sites of the enzyme
are not occupied by' the substrate molecules. So, with the increase in
substrate concentration, the rate of the reaction also increases until all the
catalytic sites of the enzyme are utilized. Rate of the reaction becomes
maximum at this point and beyond this, it remains constant.
' Nutritional Biochemistry 2) Enzymes are often inhibited by the presence of suitable inhibitors. This is
referred to as enzyme inhibition. Much of current drug therapy is based on this,
hence it is significant.

3) a) Competitive inhibition takes place when a compound having a strong I

structural resemblance with the substrate competes with it for the catalytic
site of the enzyme. Once the compound binds, the enzyme cannot convert the
inhibitor to products. Increasing substrate concentration, however, is capable
of displacing the inhibitor. Thus this type of inhibition is reversible in nature.
A good example of this is the reaction, catalyzed by succinate dehydrogenase
in the citric acid cycle. In this reaction, succinate is converted to fumarate
with the aid of this enzyme. Now, the compound malonate is structurally
similar to succinate and if present, it will compete with succinate for the
catalytic site of succinate dehydrogenase and reduce the product formation.
Thus malonate is a competitive inhibitor for this particular reaction.

b) In uncompetitive inhibition, the inhibitor only binds with the enzyme-

substrate complex making it inactive. As a result, the product formation
becomes difficult. In uncompetitive inhibition, both K, and ,
V changes.
The former increases while the latter decreases.

c) In noncompetitive inhibition, the inhibitor binds at a site on the enzyme other

than catalytic site. As there is no competition between the substrate and the
inhibitor, the inhibition cannot be reversed by increasing the substrate
concentration. For example, enzymes possessing the essential -SH group are
sometimes inhibited by metals like mercury or copper.

4) The K,,, of the enzyme for the substrate shows an apparent increase in the
presence of an inhibitor.

Check Your Progress Exercise 4

1) Coenzyme is an organic, non-protein molecule that binds with an apoenzyme to

form an active enzyme. Coenzymes can be grouped as hydrogen transferring
coenzymes and group transferring coenzymes.

2) a) Oxidation-reduction reactions
b) Oxidation-reduction reactions
c) Oxidative decarboxylation reactions
d) Transfer of carboxylic groups
e) Transfer of aldehyde and glyoxal groups.
f) Transfer of one-carbon atoms.

3) (a) - (ii)
(b) - (iii)
(c) - (i)
(dl - (iv)

4) Biosynthesis of purine and methionine is the main role of tetrahydrofolate.

5) Isozyme is an enzyme present in the same organism having different molecular

forms but catalyzing the same reaction. LDH exists in 5 possible forms.

6) a) GOT, creatine phosphokinase, LDH

b) SGOT, SGPT, alkaline phosphatase
c) amylase, lipase