UNIT 6 ENZYMES

Structure
Introduction
Objectives
General Characteristics of Enzymes
Chemical Nature
Catalytic Efficiency
Specificityof Action
Regulation of Enzyme Adivity
Classification of Enzy mes
Mechanisnl of Enzyme Action
Transition State Theory of Chemical Reactions
How Enzymes Lower the Activation Energy
Enzyme Kinetics
Concentrationof Substrate
Concentrationo l Enzyme
Effect of pH
Effect of Temperature
Enzyme Inhibition
Regulation of Enzyme Activity
Regulation by Substrate or Product
Allosteric Regulation
Regulation by Reversible Covalent Modification of the Enzyme
Isoenzy mes
Enzymes in Health Sciences

6.10 Terminal ~ u e s t i o n s
6.11 Answers
- - - - - - -

6.1 INTRODUCTION
In Unit 5 , you learnt about an important class of biomolecules, called proteins. The variety of You will recall from lJnir 5 that
functions which the proteins perform in @ie cell are crucial to life and undeniably, the most enzymes are plohular proteins.
important of these functions is carried out by a group of proteins called enzymes. An enzyme However* they differ
lot
is a biological catalyst. It speeds up or catalyses a biochemical reaction. A living cell, which
: : ~ ~ ~ ~ y Ihat
s t ~ n
we described in Unit 1 of this course, depends upon a large numbersf such biochemical
transformations for its own survival, and that of the organism of which it is a part. Since each
biochemical reaction requires a separate enzyme to catalyse it, a cell must contain a large
number of different enzymes. Acting in a co-ordinated fashion, enzymes regulate the speed
and direction of all biochemical changes. Thus, understanding enzymes and their properties is
important in understanding metabolism, which we shall discuss in Block 3 bf this course.
Therefore, in this unit you will learn about enzymes and understand their functioning in the
living cell. Besides this you will also learn that many enzymes need other nonprotein organic
molecules o r metal ions as cofactors for their proper functioning. Some vitamins and minerals
act as coenzymes1 cofactors for these biocatalysts. Most of the vitamins and minerals are
basically required to n~aintainproper cell function, growth and reproduction. We shall discuss
vitan~insand nlilrerals separately in Unit 7 of this course.

Objectives
After studying this unit, you should be able to:
describe the biological significance and general characteristics of enzymes,
classify enzymes into six major groups o n the basi.4 of their biochemical actions,

explain mechanism of enzyme action i n physico-cheiiri-1 terms, 33

 describe effect of substrate and enzyme concentration as also the role of pH and
temperature on reaction Me,
distinguish between competitive and noncompetitive inhibition,
.explain the various mechanisms of enzyme regulation,
define isoenzymes, and
describe importance of enzymes in health sciences.

6.2 GENERAL CHARACTERISTICS OF ENZYMES

Enzymes are a specialised class of proteins, and, as already mentioned, they act as
biocatalysts in the metabolic reactions. They are responsible for most of the activities that
AII the enzyme cata~ysedreactions take place within the living system and more than two thousand enzymes have been
in the liviag system are collectivelyidentified. Although enzymes are impoitant in maintaining life, they have many medical and
defined as commercial uses also. For example, determining the level of particular enzymes in blood
Metabolism consists gives a clue to the extent of heart muscle damage after a heart attack. On the commercial
and mabolk reactions. In
catabolic reactions larger molecules
side, enzymes have been used for centuries, as in fermentation to make alcoholic beverages.
arebroken~owoto~ m d u m s m a l l e r We shall now describe some characteristics of enzymes and also explain some terms used in
products and energy, whereas in this unit. .
anabolic reactiom, the e l l uses
energy to pmduce molecules which
it needs for gmwth
- and repair. Both 6.201 Chemical Nature
these series oE reactions occur
continuously in the cell.
. Enzymes, for a long time were associated with the whole living organis& with its cellular
structures intact. However, Buchner in 1897 demonstrated that enzymes were organic
substances produced by living cells and did not require intact cellular organisation for their
catalytic activity. The exact chemical nature of enzymes was established thirty years later by
Sumner. He showed that urease, an enzyme which catalyses the hydrolysis of urea, could be
crystallised and was shown to possess properties characteristic of proteins.
-.
0
11 Enzyme urease
-
H2N C - NH, + H,O 2 2NH3 + CO,
urea
This result was confirmed by Northrop and Kunitz, who crystallised several enzymes and
established that they were protein in nature. In 1960, the laboratories of Stein and Moore
Ribonuclease catalyses the established the cheinistry of the enzyme ribonuclease by deducing its amino acid sequence
ribonuc'eb add.
and a few years later, MerriWd (1969) achieved its total synthesis on the basis of i e known
amino acid sequence. This provided the ultimate proof that enzymes are no different from
other chenlicals of nonbiological origin. A complete understanding of the chemistry of an
Lysozyme is an enzyme which enzyme came with the \ ~ o r kof Philips (1965) who determined the three dimensional
degrades bacterial cell wall. structure of lysozyme. Using this information, Philips and his group proposed a chemical
mechanism for the catalytic process of lysozyme. It was for the first time. that such a feat was
accomplished. The primary amino acid sequence and the three dimensional structure of Many
enzymes is now known. This knowledge has also thrown extensive light on the general
features governing enzyme structure, function, regulation and evolution.
Proteins can be divided into two
major classes, according to one Considerable diversity of structure is seen in the enzymes. Many enzymes are simple protein
Simple molecules. This implies that the protein molecule in itself is the true catalyst. However, many
protetns, according to
lhis enzymes require the pre ence of additional nonprotein molecules for the full expression of
classification arc proteins which
produce only amino acids upon k
their catalytic function, hicb means that these enzymes are conjugated protein molecules.
hydrolysis, conjugated Let us first try to learn the meaning of some terms, which we shall be using in this unit.
proteins produe amino acids an@ Fig.6.1 gives you a general idea of these terms,.as related to the structure and function of an
other organic or inorganic. enzyme.

I substancesupon hydmlysis. -
Apoehzynre: The protein part of an enzyme molecule is known as apoenzyme.

Cobctor: The nonprotein pan of an enzyme is called a cofactor. These cofactors are
basically the additional chemical groups, which appear in those enzymes that are conjugated
You learn bbout enzyme protein molecules. Some cofactors may k (Ltnl ions such as M ~ ~ 'h ,2'v l e x organic
cohctors in Unit 7. ' molecules, such as nicotinamide adenine dinucleotide. Whatever be the nature of a cofactor,
both are required for enzyme activity.
34
 Organic molecule
I n Metal ion
activator

Active s i t e

Coenzyme
1 Substrate
Enzyme (~oloenzyme)

Fig.6.l: Schematic representation of an enzyme

Holoenzyme: The combination of an apoenzyme and the cofactor is known as the

holoenzyme:
Apoetuyme + Cofactor --+ Holoetuyme
Activator: In case the cofactor of an enzyme molecule is a metal ionsuch as, ~ e ~M$,
+ ,
Cu2+,K+,Na+, zn2+,the cofactor is known as an activator.
Coenzyme: When the cofactor of an enzyme is a nonprotein organic molecule, the cofactor is
known as a coenzyme. For example, the B group of vitamins are coenzymes that are
necessary for proper cellular respiration.
Prosthetic group: A cofactor, which is tightly bound to the apoenzyme, is generally called a
prosthetic group. However, it is possible that what is a prosthetic group for one enzyme may
be simply a cofactor for another.
Substrate: A chemical substance or substances, the transformation of which is catalysed by
an enzyme, is called its substrate. The catalytic transformation of a substrate by an enzyme
involves its prior binding to the enzyme, followed by a reaction leading to bond making or
bond breaking, resulting in a product.
--
-Active site: The part.of an enzyme where all the events of the catalytic process occur is
called its active site. Thus, it is the specific area of an enzyme to which the substrate attaches
during the reaction. Besides, the reaction is also catalysed and the products subsequently
released at this site.
The active site consists of a few amino acid side chains, some known as binding groups and
the otheras catalytic groups. The binding side chains can bind to different parts of the
substrate by hydrogen bonds and electrostatic or hydrophobic interactions. Thus, binding
amino acids can either be polar or nonpolar. However, the amino acid side chains of the
catalytic groups are of polar type only, because they bring about changes in the electronic
structure of the substrate, which is a prelude to cheniical transformations. We may mention
here that there is no clear distinction between the amino acid side chains participating in the
binding process and those which participate in the chemistry of catalysis. Some amino acid
side chains could participate in both these aspects. In Figure 6.2, we have presented a
schematic view of an active site.
chains

Active s i t e

Fig.6.2: A schematic representation of h e active site of an enzyme. The letter R with subscript shows
the position of the amino adds in the protein chain.
 --

Biomokeola 11 -
We shall now describe the efficiency of an enzyme as a biocatalyst. You will also learn that
enzymes possess enormous catalytic power.

6.2.2 ~ a t a l ~ tEfficiency
ic
Enzymes, in a living cell, perform their functions under very moderate conditions of
temperature and pH. Various chemical reactions, which are catalysed by enzymes in the
living cell under physiological conditions, would hardly occur outside the cell in the absence
of a n enzyme. For example, it would take nearly 50 years to digest a single meal without
enzymes. Another example, which we can cite, is that of the reversible reaction between
carbon dioxide and water to produce carbonic acid. This reaction is catalyseihy carbonic
RBCs are rich in the enzyme anhydrase, a n enzyme which is present in most of the tissues, especially in erythrocytes.
carbonic anhydrase They can
,
absorb COz as i t is produced i n the
body and transport it hack to tbe Carbonic anhydrase
lungs where it is released as one of CO, + H20 H2C03 ,

the products of the body. Carbonic acid

The rate of this reaction is very low, but with the presence of enzymes it is increased to about
lo7times. Another reaction, which we shall illustrate here is the decomposition of hydrogen
peroxide to oxygen and water.

2H20,
Hydrogen
peroxide
- Peroxidase
2H20 + 0,

This reaction is catalysed by the enzyme peroxidase. Hydrogen peroxide is produced as a

byproduct in many biological oxidations. It is highly toxic, and has to be decomposed quickly
to prevent any damage to the cell components. This is achieved with the help of pe'roxidase,
which affects an increase in the rate of reaction by about 101° as compared to the uncatalysed
reaction. 1
It is thus obvious that enzymes increase the reaction rate enormously, which is of utmost
significance in the biological system. Wherever it has been possible to make comparisons, a
rate enhancement of about lo4 to 1014 has been observed with the presence of enzymes in
biochemical reactions. Table 6.1 gives you some examples of rate enhancements brought
about by enzymes.
I
Table 6.1 :Comparison of nonenzplic and eny m i c catalysis I
I

Substrate Catalyst Temperature (K) Rate consdnt (k)

(mol dm-v1 sml
Amide (hydrolysis)
benzamide H+ 325 2.4 x 10-
benzamide OH 326
8.5 x
benzoyl-L- tyrosinamide a-chymotrypsin 298
14.9

Urea \ H+ 335 7.4 10-

(hydra1ysis) urease 294
5.0 x lo6

Hydrogen peroxide ~e" 295 56

(decomposition) peroxidase 295 3.5 lo7
We shall consider various factors that lead to this rate aweleratiofin Section 6.4.
The catalytic efficiency of an enzyme is expressed in terms of its turnover number. This
number indicates the nunlber of substrate molecules which are transformed in one unit of
time by one n~oleculeof an enzyme, under optimal conditions of temperature and pH. You
will learn about optimal conditions of temperature and p H in Section 6.5. We have given
turnover numbers of some enzynles in Table 6.2. As will be evident fmm this table, the rate
at which an enzyme catalyses a reaction, will vary from enzyme to enzyme.
 Table 6.2: Turnover numbers d some enymes
Enzyme Turnover number (sec")
Carbonic anhydrase 600,000
3-Ketosteroid isomerase 280,000

Lactate dehydrogenase l,o(J(J

DNA polymerase 15

An important aspect of enzynie catalysis is their specificity in acting.on a particular

substrate(s), corresponding to a particular reaction or a class of reactions. In the next
subsection, you will learn about enzymatic specificity of action.

6.2.3 Specificity of Action

An aspect of catalysis that distinguishes an enzymic from its nonenzymic counterpart, is the
specificity displayed by the former in its action on the substrate. Enzymes are highly specific
in their action, both with respect to the substrate they act upon and the kind of reaction they
catalyse. Thus, the same substrate undergoing two different types of chemical transformations
will be acted upon by two different enzymes. Physiologically the term specificity refers to the
ability o f a n enzynie to recognise and transform one particular substrate in a niixture of
several substrates. Therefore, it follows that the specificity of a n enzyme f o r a substrate not
only involves the strength of binding of the substrate to the enzyme b u t also the velocity
of the eatalysed reaction.
The degree of specificity varies from one enzyme to another. Some enzymes have a very low
specificity. This enables then1 to act on a variety of substrates, provided they contain a
particular susceptible bond such as, a peptide bond, a phosphate ester bond or a carboxylic
ester bond. These enzymes are of the degradative type, such as peptidases, phosphatases or
esterases. They generally participate in digestive processes where a narrow specificity would
be expensive for the economy of the organism There are other enzymes of intermediate
specificity such as those which display group specificities. Thus, carboxypeptidase A is
specific for the rellioval of C-terniinal amino acids containing a free carboxyl group. Another
enzyme of intermediate specificity is hexokinase which catalyses the phosphorylation of a
variety of D-hexoses. Besides the above mentioned enzymes of variable'specificity, some
enzynies show absolute specificity for a particular substrate oldy, and the best exalliple of this
type is the enzyme urease, which splits only urea. Many enzymatic reactions also display
stereochemical specificity. For example, D-amino acid oxidase is specific for D-amino acids
only and will not affect L-amino acids. As can be expected biosynthetic enzymes tend to be
highly specific so that anabolic activities are chamelised in a particular direction.
About a ce~ituryago, Eniil Fischer had proposed the "lock and key" hypothesis to explain
the specificity of enzyme-substrate interaction. Although, this hypothesis still holds good in
part, the specificity of an enzyme with respect to its substrate or a bond @be broken, is
determined by the shape of the active site, its topographical features and the proper
disposition in space of the amino acid residues participating in the process of substrate
binding and catalysis. hi the light of lliodern knowledge, o r e niay say that not oldy have the
lock and key to fit each other but they have to be flexible enough for the best fit. This
constitutes what is known as the "induced fit" theory and can be canlpared to a hand
slipping into a glove, which then causes or more appropriately 'induces' a fit. However, the
specificity is still retained as a left handed glove will not fit a right hand and vice versa. We

Most oE the enzymes are specific Eoc

,only one particular substrate. This is

Enzyme Pasihle suhstmies Only one suhstraie fie the active site witb only one particular key.
.
comanraMe to a lock. which omns uo ,

cbdn
lack

Enzymes have been classified on the basis of the type of reactions they catalyse and we shall
describe the six major classes'of enzymes which have been identified. This classification is
 Biomolecules - I1

-
have diagrammatically illustrated the "lock and key" hypothesis and the "induced fit"
theory in Fig.6.3.

Active site Active site

L 1 -

Fig. 6.3 : a) In "lock and key" hypothesis, the active site conforms precisely to the substrate
' molecule

b) In "induced fit" theory, the active site is induced to take a complementary shape by
the substrate molecule

6.2.4 Regulation of Enzyme Activity

An interesting feature of enzymic catalysis which is not shown by its nonenzymic
counterpart, is its ability to be regulated by small ions or small molecules which may be . -
substrates, substrate analogues or substances structurally unrelated to the substrate. Quite
often such regulatory molecules are the end products of a biosynthetic pathway which inhibit
the early enzymes in the pathway, thus regulating their own formation. This is o,ne of the
no st fascinating properties of enzymes, which enables fine tuning of enzyme activity in
response to changing conditions in the cell. You will learn nlore about this aspect of enzynle
function in Section 6.6.

SAQ 1
Tick [ \/ ] mark the correct statements:
a) Enzylne urease which catalyses the hydrolysis of urea was shown to be ~lo~rprotein
in
nature. 1 I
b) The combination of an apoenzyme and a cofactor is known as the holoenzyme. [ , ]
c) Specificity of an enzyme for its substrate il~volvesstrength of billding as well as rate of
catalysis. [ 1
d) The rates at which different enzymes catalyse their reactions are the same. [ 1
-
In the foregoing sections you studied the biological importance of a class of proteins, called
enzymes. We then described the general characteristics of these biocatalysts and also defined
various terms which we have used in this unit. We shall now briefly discuss the classification
and nom-enclature of the enzymes in the next section.

h.3 CLASSIFICATION OF ENZYMES

Although syste~llaticnames are used to designate the enzymes, more often collllllon nallles
are empic yed to identify them. The conlmon name of an enzyme is generally derived by
naming 'hf substrate and the type of reaction it catalyses and adding the suffix -ase to it. For
example, the oxidation of lactate to pyruvate involves the removal of hydrogen ato~nsfrom
adjacent atollls, i.e., dehydrogenation. Nicotinamide adenine dinucleotide (NAD) is the
oxidising agent used in this reaction. The enzyme which catalyses this reaction is named as
lactate dehydrogenase or LDH in short.

OH 0
I Enzyme LDH 11
CH, - CH- COO- + >-
NAD+ - CH3 - C - COO- + NADH + H+
lactate Dehydrogenation pyruvate
(substrate) (reaction type)

-
B i i k e u l e s I1
isoleucyl- tRNA synthetase, carries the systematic name, Gisoleucine: ~RNA"l e e
(AMP.fnrmino\ The c ~ r t ~ m sn-ma
t i ~ ;naii-ntar t h - t 1 ; ~ n l n . . n : ~ n;r +-
Enzymes have been classified on the basis of the type of reactions they catalyse and we shall
describe the six major classes of enzymes which have been identified. This classification is
based on the reactions they catalyse and the substrates they act on. We shall illustrate each
class of enzyme with an example.
Oxidoreductases
This class of enzymes participates in physiological oxidation- reduction processes i.e., they
catalyse electmn transfer. An example of this class is the enzyme, alcpbol:NAD
oxidoreductase, which indicates that alcohol acts as the electron donor and NAD' as the

--
electron acceptor:

Enzyme
Ethyl alcohol + NAD' Acetaldehyde + NADH
This enzyme is also known by its trivial or common name, alcohol dehydrogeqse. You
would, however, observe that the common name does not completely describe the substrates
undergoing the reaction.

This class of enzymes catalyses the transfer of a chemical group from one substrate to
another. The transferred groups cbuld be amino, methyl, alkyl, acyl, sulphate or phosphate,

-
etc. A typical example is an enzyme commonly designated by its trivial name, hexokinase.
The systematic name of this enzyme is ATP: D-hexose 6-phosphotransferase, indicating
that ATP is the phosphak donor, D-hexose is the phosphate acceptor and the transfer occurs
to the hydroxyl on 6-carbon position of hexose.
Hexokinase
ATP + D- hexose ADP + D- hexose 6- phosphate
Hydrolases
A very large number of enzymes catalyse hydrolytic reactions. Many of the digestive Pancreatic lipase isvirtually specific
enzymes, such as amylase, sucrase, lipase and all the proteases; which cause the breakdown for the hydrolysis o f primary acyl
of food materials, belong to this group. An enzyme of this group, commonly known by its groups i.e., at C-1 and C-3 positions
o f hiacylglycerol. As a result of thiz
trivial name, pancreatic lipase, degrades lipids and is known by its systematic name
2-monoacylglycerols are the main
triacylglycerol acylhydrolase. ' end products o f triacylglycerol
digestion.
/

Pancreatic lipase
Triacylglycerol + H20 2- monoacylglycerol +2 fatty acids

Lyases
'Fhese are enzymv which catalyse the elimination of chemical groups without hydrolysis,
resulting in the Pormation of a double bond. An enzyme known by its common or trivial
name, fructosebisphosphate aldoase, is an example of this type. The full systematic name of
this enzyme is D-fructose- 1,Q-bisphosphateD-glyceraldehyde 3-phosphak-lyase,
signifying that the substrate D-fructose- l,6-bisphosphate is degraded in such a way as to
give D-glyceraldehyde- 3-phosphate as a product.
Fructose-bisphosphate aldoase
D-fructose-1,64isphosphate
+ dihydroxyacetone phosphate
Isomerases
Enzymes of this class catalyse isomerisations, and include racemases, epimerases an&

-
mutases. An enzyine knowd by its trivial name,'triosephosphate ison~erase,has the systenlatic
name of D-glyceraldehyde 3-phosphate ketol-isomerase, signifying that the aldose,

Triosephosphate isomera&
D- glyceraldehyde 3- phosphate Dihydroxyacetonephosphate
Ligases (synthebes)
The function of this class of enzymes is to join together two molecules at the cost of energy
generated by the hydrolysicnf a higk energy bond. An enzyme known by its trivial name,
Bio&keules - I1
isoleucyl- tRNA synthetase, cames the systematic name, Gisoleucine: ~RNA"'l i e
(AMP-forming). The systematic name indicates that L-isoleucine is joined to
isoleucine-specific tRNA acceptor, the process being accompanied by splitting of ATP to
give AMP and py rophosphate.

ATP + G isoleucir~e+ tRNAUC +AMP + pyrophosphate + L-isoleucyl- ~RNA'~

Enzymes have also been assigned four part code numbers, The first number indicates the type
of reaction catalysed, as per the classification scheme described above. The second number
shows the subclass which refers to the substrates which participate in the reaction or the bond
attacked. The third number is indicative of subsubclass, denoting the exact specification of
the reaction ratalysed, such as the nature of the electron acceptor or the type of chemical
grouping removed. The fourth number refers to the serial number of the enzyme, in its
subsubclass. As an illustrative example, the number for lactate dehydrogenase i.5 1.1.1.27 and
for hexokinase it is 2.7.1.1.
You would note that although systenlatic names describe the enzynle system accurately, they
are otherwise, unwieldy for day to day use. Hence, old trivial names/common names continue
to be used in the biochemical literature.
SAQ 2
ldentify hydrolases from the following enzymes. Tick [ d ] mark the appropriate box.
a) Ureasc [I
b) LDH [ 1
c) Alcohol dehydrogenase 1 1
d) Amylase [ 1
Let us now discuss how enzymes increase the mte of a reaction. We shall briefly describe the
various physico-chemical factors that contribute to the catalytic power of enzymes.

6.4 MECHANISM OF ENZYME ACTION

In Section 6.2.2, you leamt about the enomlous catalytic power of enzymes, i.e., they are
responsible for a very large increase in the rate of a chenlical reaction. kl this section let us
attempt to understand the mechanism of enzyme activity. In the next section, you will learn
about the factors that influence the rates of enzynlatic reactions.
In general, the enzyme cahlysed reactions proceed through various steps, as illustrated in
Fig.6.4.

E + S =
Enzyme Substrate
ES = E S * ~ EP = E + P
Enzyme product
(1) (2) (3) (4)

Fig. 6.41 Diagrammatic representation of various steps in enzyme catalysis

In the first step the substrate (S) gets attached to the surface of an enzyme (E), forming an
enzyme-substrate complex (ES):

activated complex
 Enzyme*
In the next stage of ihe reaction, the activated complex undergoes a chemical change to form
an enzy me-product complex:

ES* -EP

The last step involves the release of the products and the enzyme is made available for further
catalysis.

You will recall from Section 6.2.1, that all the above events will take place at the surface of
the enzyme, i.e., at the active site, where the binding and the catalytic groups will be involved
in the transformation of the substrate. The most critical step in enzyme catalysis is the
formation of the activated complex and the interactions between the enzyme and substrate are
generally of noncovalent type, i.e., electrostatic, hydrophobic, hydrogen bonding, etc.
Though, in some Cases, actual covalent bonds are also formed, the interaction between an
enzyme and a substrate has to be weak enough, so that enzyme-product complex can break
apart to release the product and thus regenerate the enzyme.
We shall emphasise here that the astronomical rate enhancements associated with enzymes
are not a magical effect, but a phenomenon, which can be understood qualitatively in
accordance with the principles of physical and organic chemistry. It is also generally
accepted that usual reactions such as nucleophilic, electrophilic, homolytic reactions and
rearrangements etc. are invol;ed in the transformation of the substrate in enzymatic reactions.
Also the efficiency of enzymatic reactions has been accounted for by suggesting factors like
proximity effect and orientation effect. However, the precise quantitative contribution of each
of the physico chemical factors to final rate enhancement in any particular enzyme is still a
matter of guess only.
Let us now discuss, in simple terms, the principles underlying catalytic power of enzymes.

6.4.1 Transition State Theory of Chemical Reactions

A chemical reaction can occur if it is thermodynamically feasible. This means that the
conversion of reactants to products must result in a negative free energy change, i.e., AG

I must be negative under the existing conditions, such as the concentrations of reactants and
products (Fig. 6.5).
AG of activatic (~6

-
Reaction Coordinate
Fig.6.5: Change in Pree energy level during the progress of n reaction Prom reactants to products

As is evident from the reaction coordinate, the reactants have to overcome a free energy
barrier before the products can be fonned. This energy barrier is known as the activation
energy (E) of the reaction. The molecular structure and conformation corresponding to the
peak position in this profile is called the transition state of the reaction. In the transition
state the chemical bonds are in the process of being formed or broken. It is, therefore, the
most activated and hence highly unstable entity in the reaction pathway.
The concept of the transition state helps to express the rate of reaction, in terms of the free
energy of activation. Since the molecules in the transition state are the ones which lead to the
formation of products, the rate of reaction will depend on the concentration of molecules i n
Biomoledes - I1
the transition state. For this concentration to increase, with a corresponding increase in
reaction rate, the free energy of activation has to decrease (Fig. 6.6). For this to happen, the
transition state may be reached with a smaller expenditure of heat or enthalpy of activation
AG - AH-TAS 3 3.
( A H or a smaller decrease in the entropy of activation ( A S This is precisely what

(a) No enzyme pre.sent (h) Enzyme present

Fi. 6.6: Effect of enzymes on the activation energy. T h e reactant molecules have to get over the
energy h a r r i e r to form the products.

enzynles achieve and thus bring about a rerlarkable increase in the reaction rate. It should lx
clear from Fig.6.5 and Fig. 6.6, that enzymes only decrease the free energy of activation, so
that Illore and 111oreof reactant l~~olecules
pass the energy bamer to fonll the products. Thus,
the enzynles donot change the equilibrium point of the reaction which is related to the free
energy of the reaction (A G ;Fig. 6.5) but merely speed up the rateat which the equilibrium
point is reached.

We shall now discuss in qualitative tenlls how e~~zyllles

cause a decrease in energy of
activation. -
6.4.2 How Enzymes Lower the Activation Energy
A chelllical reaction results fro111random collisions between reacting ~ ~ ~ o l e c u lThis
e s . nleans
the reactants have to collie very close to each other. In a nonenzy~naticreaction, the
probability of such a n event is low. Even if the reactants collide, most of the collisions are not
effective, i.e., they do not result in a chemical reaction. Only those collisions will be effective
and yield products where the reactant n~oleculeshave adequate energy and their participating
groups in the reaction are properly oriented with respect.to each other. However, such a
effective collision is a rare event and occurs with a low probability. Further, the proper
orientation of groups causes a decrease in the entropy of the system. This decrease in entropy
contributes to high values for free energy of activation, which explains the low reaction rate
of a nonenzynlatic reaction.
An enzyme on the other hand possesses an active site which binds the interacting molecules
and thus brillg the111close ellough to react (in effect collide). This is known a s the proximity
effect. As a result of this effect extren~elyhigh concentrations of reacting nlolecules are
attained, resulting in a large increase in reaction rates (in effect, increase .in collision rates).
Thus, e l ~ z y n ~ "collect"
es substrates fro111 the reaction ~llediunland "make the111 to collide".
By constructiag 111odelorganic c o m p o u ~ ~ dins which two iatencting g o u p s were attached to
a single molecule, the proximity effect of an enzyme has been mimicked. It h.as been
demonstrated that a rate increase of abdut lo4 fold can b e realised by this factor alone.
The other corldition of a fruitful collision, nanlely the proper orientation of the reacting
groups, is also realised by the enzymes. The active site of an enzyme with the. help of its
binding groups, holds the interacting molecules in a correct rigid orientation with respect to
each other. This is known as the orientation effect. Thus, substrates are precisely oriented at
the active site and hence properly positioned for reaction (in effect collision). This effect has
also been imitated and a rate increase of lo4 observed. Thus, a total rate enhancement of lo8
results from proxin~ityand orientation effects alone in enzyme catalysis.
With these effects an enzyme overcomes the unfavourable entropic barrier, which is
characteristic of nonenzymic reactions. However, the enzyme pays a price to overcome the
loss of entropy and it is the bindi~lgenergy of enzynle- substrate interaction at the active site,
which is used to yay for this price. It is thus easy to understand how the enzytne decreases the
free energy of activation of a reaction.
Another contributing factor to ellzynle catalysis is the "induced fit", which we nle~ltio~led in
Enzymes I!
subsection 6.2.3. This suggests that when an enzyme-substrate complex is fornled, the
conformations of both the enzyme as well as the substrate change. This conforn~ationchange
in the substrate produces a strain ia the fornl of distortio~lof bond angJes and ho~ldle!l&!)!Jhs
which brings it closer to the transitio~lstate. This, in turn, reduces the energy of activation
required for converting a substrate into its products. We may mention here that using model
systems, it has been shown that subjecting a substrate to strain can increase its rate of reaction
by a factor of 10'.
Lastly, the catalytic functional groups at the active site also contribute to rate enhancement.
YOUwill recall that many organic reactions are catalysed by H* or OH-ions. However, in a
biological medium, with the exceptio~lof gastric secretio~ls,the coacentration of these ions is
very low. At the active site of an enzyale, various acidic or basic groups acting as catalytic
groups can act as proton donors or acceptors. They thus effectively catalyse a biochen~ical
reaction, as their simultaneous action on the substrate can be much more significant than the
chance encollnter of a reactant with all acidic or a basic group ill the ~lonenzy~llicreaction.
We can su~nmarisehere that enzytlles are highly effective catalysts Ixcause they brjng
together interacting nlolecules in a pmper orientation. Also the functional groups at the active
site pmvide pmton donors and acceptors in high local concentration. These groups are also
properly positioned to bring about a reaction.
You can check your understanding of enzymes by attempting the following SAQ.
SAQ 3
Fill in the b l a n k with appropriate words.
a) E ~ l z y ~ l l...........................
es the rate at which equilibrium is attained in a
reaction.
b) Enzymes ......................... the activation energy of a reaction.
c) Ellzytnes overcolne the entropic b a m e r to chenlical reaction by ...................
a n d . .............................effects.
d) I n the ........................... state, the chemical bonds are in a process of
being fonned o r being broken.
In the foregoing section we explained how the enzyme nlolecule is able to bring about a rate
enhancement. You will now learn about the kinetics of enzymatic reactions. We shall
describe the factors that have a direct effect on the rate d enzymatic reactions.

6.5 ENZYME KINETICS

'
E ~ l z y n ~kinetics
e deals with the rates of enzynlatic reactio~lsa ~ the
d various parallleters that
govenl these rates, such as concentration of substrate, c o ~ l c e ~ ~ t r a of
t i oeluynle,
~l pH,
temperature and the presence of various substances that may be inhibitors or activators. Study
of enzyme kinetics is important because it throws some light on the mechanisnl of action of
a n enzyme. Such a study also provides infornlation a n the behaviour of an enzyme in the
cellular eavironnzent and gives us a clue regarding the regulatory mechanisn~savailable to the
organisnl for fine control of ellzynle activity.
Before we describe the factors which influence reaction rates, let us briefly discuss how
r e a c ~ i mrates are measured.
Measurement of Reaction Rates of Enzymes
'.
The activity of a n enzyme can be nleasured by monitoring the tilne- dependence of the
chemical change that occurs during an enzymatic reaction. The enzy lne is incubated with the
substrate under o p t i ~ ~ ~conditions.
un~ The reaction proceeds with the disappearance of the
substrate and the formation of a new product. These changes are monitored by withdrawing
snlall aliquots and analysing then1 for the forn~ationof tbe product o r disappearance of the
substrate. I n some cases such measurements can be made directly on the incubation mixture
without the necessity of withdrawing aliquots. For example, in several oxidation-reduction
reactions, where NAD o r NADP is the electron acceptor, the progress of the reaction can be
continuously monitored by following the reduction of NAD (or NADP) to the reduced
product NADH (or NADPH) which absorbs at 340 MI. Same principle is applied to monitor
nlany other enzyme catalysed reactions, where either the substrate or the product has a unique
and easily measurable absorption spectrum.
 -
B i o m o l d e s 11
Let us now learn how concentration of substrate can alter rate of a biochemical reaction.

6.5.1 Concentration of Substrate

In order to outline the effect of substrate concentration on reaction rate, we shall discuss the
reaction between a single substrate and an enzyme for the sake of simplicity. If we consider
an enzyme as a reactant, then this is equivalent to a chemical reaction between two chemical
The condition of initial reaction
substances i.e., enzyme and its substrate. However, there is an important difference between
rate is important because it ensures
that very little product is present to
enzymic and nonenzymic systems with respect to the dependence of rate on the reactant
initiate the reverse reaction, which concentrations. For example, if we carry out a nonenzymic reaction between two reactants A
may complicate results. and B, keeping the concentration of A constant and changing the concentration of B, then the
initial rate of product formation will be directly proportional to the concentration of the
reactant B. If on the other hand the conditions of an enzymic reaction are so arranged that the
enzyme concentration is kept constant and the concentration of substrate is changed, then the
initial reaction rate varies hyperbolically with increasing substrate concentrations. The rate
reaches a maximum velocity at high substrate concentration which remains uneffected by
further increase in substrate concentrations. This is illustrated in Fig. 6.7.

I I Maximum Rate ( V m 3

Rate is unaffected by further

increase in substrate
concentration in this region

- Rate is directly proportional

to substrate concentration in

-
Concentration of substrate
Fig. 6.7 : Variation of reaction rate with substrate concentration keeping enzyme concentration
constant

The fact that in an enzymic system, as opposed to a nonenzymic system, the reaction velocity
reaches a saturating value at high substrate concentrations can be explail~edas follows. In a
nonenzyiiiic reaction, the reaction rate is dependent on the nur~lberof effective collisions
between the reactants. The number of such effective collisons would increase in direct
proportion to the concentration of one of the reactants, if the concentration of the other
reactant is kept constant. On the other hand, in an enzymic reaction the effective collision
between the enzyme and the substrate leads to the forn~ationof an enzyme-substrate complex,
in which the substrate is finilly bound to the eilzyltie at its active site. This complex then
breaks down to give the product (P) and releases the original enzyme. Since the number of
active sites is limited by the enzyme concentration (held constant), the reaction rate will
.increase with substrate concentration only till all these sites are filled. Under these conditions
the system will give maximum possible rate of reaction. Thereafter, there will be no further
increase in the rate of reaction.
The shape of the curve as shown in Fig. 6.7, depicting the dependence of reaction rate on the
substrate concentration, is a hyperbola. Michaelis and Menton were the first to recognise this
relation for enzyme catalysed reactions and put the same in a mathematical form in 1913.
This equation for enzytue kinetics is known as the Michaelis-Menton equation :

Rate of reaction - v
",
,[Sl
[SI + K m

Where [S] is the molar concentratien of the substrate and V , is the maximum possible rate
for a given enzyme concentration. K,, also called Michaelis constant, is numerically equal to
the concentration of the substrate at that stage when the dbserved a t e of the enzyme
1
catalysed reaction is one half of the maximum rate i.e., v = - V,. This will be clear to you
2
from Fig. 6.8. From the above equation, it is clear that rate will tend to acquire a l~laxiiilui~~
value when IS]is very high.
 Initial rate

-
Concentration of substrate
Fig. 6.8 :Km is equal lo lbe concentralion of ibe subslrale reqnircd lq give an initial reaction rate
corresponding lo balf of Vm..

We shall now briefly explain what Vma,and K, convey in enzyme kinetics.

Sigrliticance of V,
At V,, all the enzyme molecules have formed enzyme-substrate complex (ES) and are
continuously catalysing the conversion of substrate into the product. Thus at V, value the
enzyme is fully saturated. V, values can be used to compare the activity of various
enzymes, if they happen to catalyse the same reaction.
Sigolficance of Km
1
As we have mentioned already, Km is equal to the concentration of the substrate at - V,,,.
2
Since at V, all the enzyme molecules have formed enzyme-substrate complex, it, therefore,
follows that the concentration of substrate (K,) required to convert hall of enzyme molecules
to ES complex, is a measure of the affinity of the enzyme for substrate. A snlall value of K,
signifies the high affinity of the enzyme for the substrate, since r low concentration of
substrate would be needed to saturate the enzyme. Similarly, a large value for Km would
indicate.a relatively high concentration of substrate for saturating the enzyme, thus signifying
a low affinity of the enzyme for its substrate.
You have thus learnt that increase in substrate concentration has a corresponding affect o n
the rate up to a certain point, beyond which the rate remains unaffected to any further
increase in concentration of substrate. We shall now discuss the role of enzyme concentration
in the kinetics of enzymic reactions.

6.5.2 Concentration of Enzyme

In enzymic reactions, the enzyme concentration is generally very low, as compared to the
substrate concentration. The rate, therefore, is proportional to the concentration of the
enzyme, provided a purified enzyme is used and substrate concentration is very high. Under
these conditions, for example, if enzyme concentration is doubled, the rate of conversion of
substrate to product shows-a corresponding increase (Fig. 6.9).

concentration is

-
Enzyme c~ncentration -
Fig. 6.9 : Variation d rate o t cntalysed reaction wlth concentratiaa or the enzyme

Let us now discuss how catalytic activity of a n enzyme is affected by the p H of the solution
in which the edzyrnic reaction occurs.
6.53 Effect of pH
i
An enzyme catalysed reaction is strongly influenced by the hydrogen ion concentration, i.e.
p H of the reaction mixture. Usually a small change in pH causes an appreciable change in the
ability of an enzyme to function as a biological catalyst. The curve showing rate of enzy~tte
catalysed reaction against change in pH of the reaction mixture is generally bell-shaped
(Fig. 6.10). You would observe that enzyme activity is maximum only in a narrow pH range
and decreases at both higher and lower pH.
I
Maximum catalytic

Reaction rate
I
Incre
Fig. 6 1 0 : Effect of pH on tbe activity of M enzyme

The pH where an enzyme shows rttaximuni reaction rate is known as its optimal pH. Most
enzymes have a maximum catalytic activity around pH 7, which happens to be the pH of
most of the biological fluids. However, many enzymes do have maximumactivity at higher
o r lower p H than this. An important example is that of pepsin, which is a digestive enzyme of
the stomach. It has maximum activity around pH 15,which is the pH of gastric juice. We
have listed optimal pH values of some enzymes in Table 6.3.
Table 6.3
Enzyme Optimal pH values of some
enzymes
Pepsin 1.5
a:Glucosi&se 5.4
Urease 6.7
a-Amylase 7.0
Carboxypeptidase 7.5
\
Trypsin 7.8
Alkaline phosphatase 9.5
In qualitative terms, this effect of pH on enzyme activity can be attributed to several factors,
some or all of which may operate at the same time. The change in pH can cause denaturation
of the enzymic protein, rendering it inactive. Further, you may recall from subsection 6.2.1
that active site of an enzyme consists of amino acid side chains which font1 binding and
catalytic groups. The catalyticactivity may be possible only when these side chains are in P
correct state of ionisation. The state of ionisation of these side chains would in turn depend
on the pH of the reaction mixture, thus influencing enzyme activity. We will illustrate this
with an example. Let us suppose an enzyme needs two chargedlionised side chains (--Nq
and-COO-) at its active site for catalysis. This ionisation state would be possible only in a
panicular pH range, corresponding to the optimal pH:

Unionised

Below the optimal pH (decreased Above the optimal pH

or no catalytic activity) (decreased or no catalytic

As you would observe from the above representation, at acidic p H (lower pH value than
optimal pH)the - COO- group gets protonated to - COOH and at basic pH (higher pH than
optimal pH), the - N K group gets deprotonated to - NH2. Consequently in both these
situations the catalytic activity decreases for lack of proper combination of the ionisable side
chains in the active site. Lastly, the effect of pH on enzyme activity could also result from a
change in ionisation state of a charged substrate with change in pH, affecting its binding to
the enzyme.
You will now learn about the fourth factor which influences the rate in an enzyme cataly$ed
&action.

6.5.4 Effect of Temperature

You will recall that increase in temperature leads to increased frequency of collisons between
reactants. Thus rates of chemical reactions increase with increase in temperature. However, in For most of the enzymes optimal
tem~eraiu~sareatorahovetboseof
the case of enzymatic reactions this increase occurs only within a narrbw temperature range the alls in which they occur.
beyond which !he rate decreases sharply, suggesting that the enzyme may have an optimal
-temperature, where the rate is maximum as shown in Fig. 6.11.

Optimal temperature

Reaction

Temperature ---,
Fig. 6.11 : Effect of temperature on the activity of an enzyme

As you would have observed, enzyme activity increases with temperature. This, evidently, is The activity of most e f ibe enzymes
due to increase in the number of collisions between the enzyme and substrate molecules, and is usually destroyed by heat.
also doe to an increase in the energy of these collisions. With further increase in temperature, However* are Ihe
enzymes of bacteria, living in hot
enzyme activity decreases sharply. The latter is due to the denaturation of the enzyme springs at temperatures of 60-80°C.
proteins with heat. We can thus say that though reaction rate increases with temperature, the
rate of inactivation of the enzyme also increases simultaneously. The concept of optimal
temperature for enzyme activity does not seem to be a satisfactory one, since it is the result of
an increase in enzyme activity due to increase in temperature, and loss of activity due to
inactivation of the enzyme. Since the latter factor is time dependent, the optimal temperature
will itself depend upon the time taken for rate determination at a particular temperature. The
change in rate of reaction for any 10" rise in temperature is known as its Qlo, or temperature
coefficient value. This value is close to 2 for most chemical reactions. For, enzymatic
reactions on the other hand the Qlo values are lower.

In the above subsections you learnt about the factors which effect enzyme activity. We shall
now describe the enzyme inhibitors, which can decrease the rate of enzymatic reactions.

6.5.5 Enzyme Inhibition

Enzyme activity is inhibited by several factors, such as unfavourable pH conditions, rise (or
sometimes fall) in temperature or presence of protein precipitants, such as alcohol, acetone
and trichloroacetic acid. Inhibitory action, caused by these agents is of a general nature a&
nonspecific. Of greater interest in terms of structure and function of enzymes, are inhibitors
known as competitive and noncompetitive inhibitors. '
Competitive inhibitors have a structure resembling that of the substrate. The structural
similarity between the substrate and the inhibitory compound enables the inhibitorlo compete
with the substrate for binding to the active site of the enzyme. This prevents the access of the
substrate to the active site and the formation of ES complex. Since the substrates of enzymes
are generelly metabolites, participating in metabolic transformations in the cell, competitive
inhibitors are known as antimetabolites. One of the best known examples of a competitive
inhibitor is sulphanilamide. This is a competitive inhibitor of P-amino benzoate, a compound
utilis~din the synthesis of folic acid coenzymes essential for the transfer of one carbon
fragments. It is, therefore, easy to understand why sulphanilamide is successfully used as a
drug, inhibiting the growth of bacteria. La fact many other drugs also work by acting as
competitive inhibitors in enzymatic reactions necessary for the growth and survival of a large
number of bacteria. 47
 p-Amino benzoic acid Sulphanilamide

Another well known competitive inhibitor is malonic acid, which inhibits the oxidation of
succinic acid to fumaric acid by the enzyme succinic dehydrogenase. The competitive action
of malonic acid against succinic acid can be appreciated by a comparison of their structures,
which are closely similar.
COOH COOH
I I
CH2 CH2
I I
COOH CH2
I
COOH
Malonic acid Sucanicacid

It is possible that the active site of the enzyme mistakenly accepts ~nalonicacid instead of
succinic acid as its substrate, causing co~npetitiveinhibition. You will find a diagrammatic
representation of competitive inhibition in Fig. 6.12.

Enzyme Enzyme-inhihtor complex Enzyme Enzyme-substrate complex

(a) E t I = El (b) E t S = ES
Fig. 6.12 : a) Formation of an enzyme-inhibitor complex
b) By increasing substrate concentration, ES concentration gets increased and EI
concentration decreases which reverses the effect of the inhibitor

A characteristic feature of competitive inhibition is that it can be reversed by increasing the

substrate concentration. We can detect competitive inhibition by a study of the rate of
enzymatic reaction in the presence, and in the absence of a competitive inhibitor. You can
In absence of a
competitive inhibitor V,,, is unchanged
J

In presence of competitive

Substrate concentration
Fig. 6.13 : When a competitive inhibitor is present, Vonx remains unchanged but K n is increased
observe fro111Fig. 6.13 that V,,,, remains unchanged in the presence of a competitive
inhibitor, whereas K,, is increased, since a higher substrate concentration would be needed to
out-conlpete with the inhibitor and to achieve the saturation of half of the enzyme nlolecules.
Noncompetitive inhibitors inhibit enzyme action by combining with a group essential for
the activity of the enzynle o r by removing a metal ion involved in the activity of the enzyme.
These compounds act by converting the enzyme into an inactive or less active form. Such
inhibitors cannot be displaced by excess substrate and hence are called non conlpetitive
inhibitors. Since their action does not involve displacenlent of the substrate, they have no
effect on the K, of the substrate. However, since the inhibitor makes the enzyme less active,
Vma,is'affected.
SAQ 4
Tick [ \/ ] mark the correct statement.
Increasing the tenlperature of an enzynlatic reaction is accompanied by
a) activity remaining constant 1 1
b) activity i~~creasiilg
coiltilluously [ 1
c) activity decreasing conti~~uously [ 1
d) activity first illcreasiilg and then decreasing [ ]
SAQ 5
Tick [ \I ] mark the appropriate statement.
A competitive inhibitor of an enzynie acts by
a) modifying one of the amino acid residues of the enzyme [ 1
b) modifying structure of the substrate [ 1
c) competing with the substrate to bind to the active site of the enzynie [ ]
d) increasing temperature of the reaction nlixture [ 1
We have described the various factors that alter the rate of an enzynlatic reaction, and you
studied about enzynle inhibitors also. You will recall that enzymes bring about enornlous
changes in the rates of biochenlical reactions. This makes life possible for an organisnl.
However, their importance for a livii~gsystern is not only linlited to increasing the rates of
biochelrlical reactions. Their activity can also he controlled o r regulated. This is highly
significant in a living systenl, for rates of individual biocatalytic reactions can be controlled
and con~binedinto different metabolic pathways. These individual itetabolic pathways are in
turn integrated into an overall nletabolic systenl in the living organisnl. Let us now discuss
more about the regulation of enzyme activity in the following section.

6.6 REGULATION OF E N Z Y M E ACTIVITY

-
Lehninger defined the living cell as a self assembling, self adjusting, self perpetuating
isothermal system of n~oleculesthat exchanges matter and energy with its environment. The
cell accomplishes this by a process called metabolism, in which many consecutive chemical
reactions are organised into metabolic pathways. You will recall that nletabolisnl consists of
anabolism and catabolism. The term anabolisni refers to those pathways of nietabolisni in
which low molecular weight precursors are transformed into inore col~iplexsubstances which
include carbohydrates, lipids, nucleic acids and proteins, leading to the synthesis of llew
cellular con~ponentsand overall growth. Catabolis~r~ on the other hand is the process by
which complex and siniple substances are broken down in other lnetabolic pathways to
produce energy, either in the fonn of heat or in the fornl of high energy compounds. Both the
anabolic and catabolic pathways are interconnected.
It is self evident that such r complex system of biochemical transfor~nationshas to be so
regulated that concentrations of certain key n~etabolitesare controlled, both in tenrls of tiirle
and space, to direct lnetabolism in a desired direction, and not allow it to drift into the
ultimate products of aerobic ~rietabolismi.e., carbon dioxide and water. Since ~netabolislnis
driven by enzymes, regulation of metabolism necessitates regulation of enzytlle action. We
shall now look at some of the principal mechanisms by which enzyme activity is regulated.
 -
Biomolecules I1
6.6.1 Regulation by Substrate or Product
The intracellular concentration of a substrate can sometimes regulate the action of the
corresponding enzyme. Such regulation is possible when the K,,, value of the enzyme is much
higher than the intracellular concentration of the substrate, so that enzyme activity is first
order with respect to substrate concentration.
The product of an enzynlatic reaction is expected to have a structure resenlbling that of the
substrate and may act as an inhibitor of the reaction, when it accutnulates in sufficient
concentration. However, the significance of such product inhibition in enzyme regulation
cannot be very high since one would expect the product to accumulate to a high concentration'
before significant inhibition is achieved. Also it is difficult to conceptualise the control of a
metabolic pathway, if the product of one particular enzyme inhibits that enzyme
independently of the needs of the whole pathway.
An iluportant biological tllechanist~lwhich controls enzyme activity is allosteric regulation.
This interaction may bring about inhibition or stilnulation of activity by altering the activity
of key enzymes. This is possible when enzytues interact with the molecules produced in the
cell. Let us explain more about this type of regulation.

6.6.2 Allosteric Regulation

Regulation of some of the biosynthetic pathways occurs by this mode. In such cases, the Brst
enzyme of a biosynthetic pathway is strongly inhibited by the end product of that pathway
(also called feedback inhibition). For example, aspartate carbamoyl transferase, the enzyme
which catalyses the first step in pyritnidine biosynthesis in E. coli, is inhibited by the final
product of this pathway, which is CTP, a pyrimidine nucleotide (Fig. 6.14).
aspartate N-Carhamoyl-L-aspartate
L-Aspartate + Carbamoylphosphate -2
carbamoyltransferase + Orthophosphate /

UTp UMP
j
End product o € pyrimidine /
hiosynthesis in E.Coli
Fig. 6.14 : Inhibition of the first enzyme by the end product of the pnthwny

Interestingly ATP which is a purine nucleotide, activates this enzyme (Fig. 6.15). Thus, as a
result of the opposing action of these two types of nucleotides, production of purine and
pyrimidine nucleotides is properly balanced for the synthesis of nucleic acids. On the basis of
f ATP

of CTP
(inhibition)

I/ / 1 In absence of any

(L-Aspartate) (rnmol dm -')

Fig. 6.15 : ErCcct of CTP and ATP molecules (regulntors) on the kinetics of aspartate
cnrbnmoyltrnnsfernse from E. C d i

work carried out on several other pathways, the following general conclusions regarding this
type of regulation have emerged.
Only the first enxyme of the biosynthetic pathway is affected, due to the feedback
inhibition by the final product of the pathway.
This final product of the metabolic pathway bears no structural resembla~lceto the
substrate or product of the first enzyme, thus, providing a tnode of enzyme regulation
i~ldependeiltof the substrate or product (also refer to subsectiotl 6.6.1).
 Enzymes
The regulatory molecules, also called effectors, being structurally different from the
substrate or product of the first reaction, do not bind to the active site of the first
enzyme, but would perfonn their regulatory function by binding at another site on the
enzyme, called the allosteric site. This bitldiilg is reversible. Enzymes so regulated are
called allosteric enzymes.
1.

Such allosteric'enzymes do not exhibit normal Michaelis-Menton or hyperbolic

kinetics. In such cases, the plot of velocity against substrate coilceiltratioil shows
sigmoidal behaviour (Figs. 6.15 & 6.16). This behaviour indicates that at certain
concentratioils of the substrate, the eilzyine activity is inuch more iilflueilced by
changes in conceiltration of the substrate, than would be the case for an enzyine
characterised by normal kinetic behaviour. Sigmoidal kinetics of a regulatory enzyme
can be changed to hyperbolic kinetics by subjectiilg the eilzyine to physical or
chemical treatment (Fig. 6.16).

Treated enzyme

v Untreated enzyule
(sigmoidal behaviour)

0 10 20 30
(LAspartate) (mmol dm " )
Fig. 6.16 : Conlparison of kinetics of the treated and untreated regulatory enzymes, aspartate
carbamoyltransferase from E.Coli

Regulatory enzymes generally posses an oligomeric structure like haemoglobin

(subsection 5.4.6) which is an oxygen transport protein of blood. The multiple
subuilits which coilstitute the oligoineric structure of these enzymes, are held together
by weak iloilcovaleilt forces. The binding of the regulator lnolecule to such an eiuyine
at the allosteric site brings about a reversib!e coilfonnational change i.e., a change of
shape in the eilzyine subunit, causiilg a change in the structure of the active site
(Fig. 6.17), leading to alteration in enzyine activity.

,Reeulator molecule

~ e ~ u l a t o i allosteric
site
or '
Active site.

Fig. 6.17 : Diagrammatic representation of the reversible structural change in a regulatory enzyme,
brought about by a regulator

You will recall that in the case of haemoglobin, a plot of percent oxygen saturation against
oxygen pressure is sigmoidal in nature (Fig. 5.9). The sigmoidal kinetics of regulatory
eilzyilles denotes interaction between subunits, so that changes in the active site structure of
the first subunit would affect active site structures of other subunits through rearrangement of
noncovalent interactions. Such interactions between subunits, in response to the binding of a
regulator molecule to the allosteric enzyme, are called cooperative interactions.
Regulation of enzyrne activity by conformational changes in the enzyme, induced by the
binding of regulator to its allosteric site, is possibly the 111ostimportant ineans of nletabolic
regulation or control. Another mechanisnl by which enzyme activity is regulated, involves
reversible covalent t~~odification of the enzyme. Let us understand this mechanisnl in the next
subsection.
 -
Biomolecules I1
6.6.3 Regulation by Reversible Covalent Modification of the Enzyme
Several enzymes, crucial to biosynthetic and dewdative pathways, are regulated by
reversible covalent modification of their side chains. Tbese enzymes are present in two
different forna, possessing different catalytic efficiency. These forms are interconvertible by
the action of other enzymes, some of which catalyse the modification of the side chains,
whereas others catalyse reversal of this modification.
We shall illustrate this with two enzymes, phosphorylase and glycogen synthase. These
enzymes are involved in the degradation and biosynthesis of glycogen, respectively. The
enzyme phosphorylase catalyses the following reaction:
(Glycogen), + orthophosphate W (Glycogen), - ,+ a - D - glucose 1 - phosphate
The enzyme phosphorylase has two forms. The active form (a) and the inactive form (b). The
only structural difference between the two forms is that side chain of serine, at position 14 in
the sequence of the enzyme, is phosphorylated in the form a but not in the form b. This
phosphorylation of the enzyilie is catalysed by the enzyme phosphorylase kinase, in presence
of ATP and ~ g ~The + reversal
. of this process i.e., the conversion of phosphorylase a to
phosphorylase b is accoiiiplished in the presence of another enzynle, phosphorylase
phosphatase, which catalyses the reinoval of the phosphoryl group. These reactions are
schenlatically represented in Fig. 6.18.

ATP ADP

Phosphorylase (inactive) Phosphorylase a (active)

a Enz-Ser OH

Orthophosphaie
Enz-Ser

Fig. 6.18 : Schematic representation of the interconversion of phosphorylase o and phosphorylase b

Similarly the other enzyme, glycogen synthase, which catalyses the synthesis of glycogen (as
opposed to phosphorylase, which catalyses its breakdown), is converted to an inactive form
by phosphorylation, a process which also leads to the activation of phosphorylase. It is thus
clear that the sinlultaneous activation of phosphorylase and inactivation of glycogen synthase
in a reversible manner, are complementary processes that allow a well integrated control of
glycogen nletabolisnl.
Reversible conversion of active to inactive enzyme fornls is a potent mechanism of enzyme
regulation. The amounts of the active and inactive forms of the enzynle increase or decrease
rapidly, because their interconversion is enzyme catalysed. This also results in a large
amplification of the initial signal, where a series of modifying enzymes act in succession to
proniote a final metabolic event. The continuous activation and inactivation of the enzyme
also enables the enzyme system to be more responsive to metabolic requirements.
SAQ 6
Tick [ 4 ] mark the following statements as true or false.
a) Allosteric effectors fu~ictioiiby billding not to the active, but to an alternative site on the
eluy me.
[True/False]
b) Enzyliie phosphorylation can result either in a niore active or a less active enzyme.
[TrueFalse]
c) I11 the feed back ltlechanisln the elid product of the nletaholic pathway does not inhibit
the first enzyme of the pathway.
[True/Falsc]
d) Reversible interconversion of an active enzyme to the inactive form does not regulate
enzyme activity.
[TrueFa Ise]
You will now briefly learn that some enzymes can exist in more than one forni, which differ
in their kinetics, as well as regulatory properties. Genetic factors are responsible for these
different forms, and ehch form is tuned to a function in a specific tissue.

ISOENZYMES
Sometimes different fonns of.an enzyme, catalysing the same reaction, occur in a species.
They are called isozymes or isoenzymes. They may arise from minor nlodification of the
same amino acid sequence or they may have different amino acid sequences. These changes
are genetically determined. T h e t e r m isoenzyme is reserved for those forms of a n enzyme
which catalyse the same reaction, a r e isolated from the same species a n d differ in amino
acid sequences because of genetic reasons. The isoenzymes generally differ in their kinetic
and regulatory properties and their distribution is tissue specific. This suggests that the
kinetic and regulatory properties of a particular isoenzyme are attuned to the metabolic
requirements of a particular tissue, where it is found in abundance. The nomenclature of the
isoenzymes is based on their electrophoretic mobilities towards the anode. Thus hexokinase
isoenzymes are numbered I to IV, with isoenzyn~eI having the lowest mobility. Isoenzynles
of lactate dehydrogenase represent an interesting case of the origin of isoenzymes. All
isoenzymic forms of this enzyme consist of four subunits. These subunits are of two different
types, a and B. These combine in five different ways viz. a,, a$, a2p2,ap3and B,, to
respectively produce five different isoenzymes, LDH-1 to LDH-5 (Fig. 6.19). LDH-1 has the
highest electrophoretic mobility towards anode. LDH-1 is predominently present in heart and

-
LDH-5 is mostly present in skeletal muscle and liver. These isoe~lzynlesdiffer both in their
heat lability and sensitivity to i~lhibitiodby excess \~~bstrate.

-
-
LDH-1 (a4)
II - - - --- ----- (+)
A

LDH-2 (a3 B)

- - - -
--
LDH-3 ( a z Bz)

LDH-4 ( a 83) - -
LDH-5 (84) -----

Fig. 6.19 : Electrophoretic mobility of isoenzymes of lactate dehydrogenase

Several enzymes such as alkaline phosphatase, hexokinase, amylase, and glucose 6-phosphate
dehydrogenase, exist in isoelizynlic fonns, which are, however, not as exhaustively studied as
those of lactate dehydrogenase.
SAQ 7
Tick [ d ] mark the niost appropriate statenlent.
Isoenzy~tlesare:
a ) e ~ e d r o ~ h o r e tvariants
ic of the same enzyme.
b) electrophoretic variants produced by modification of side chains.

d) different enzymes acting on the same substrate

-
In this unit we ~iiainlydiscussed the rolc of c~izyniesas biocatalysts, regulating ~lletabolic
pathways in the organism. However, you liiay recall fro111 Section 6.2 that enzynies have
many co~nmercial,as well as niedical uses also. We will attelllpt to givc you sollie idea of the
significa~ireof enzyiiies as iniportant "tools" in nledical science. Let us, therefore, give you
a brief account of their role in health sciences.
6.8 ENZYMES IN HEALTH SCIENCES
I n the present times enzymes are used to diagnose and to treat diseases. Enzymology is an
essential part of the day to day life of modem clinicians. The diagnostic value of certain
enzymes arises from their differential distribution between the blood plasma and cells of
other tissues. For example, the enzynles involved in blood coagulation are found exclusively
in the plasma. On the other hand, many other enzynles are present in much higher
concentrations in the tissue cells, than in blood. These are released into the blood and various
biological fluids only when there is routine destruction of the cells. Their non~iallevels in
plasnla are insignificant, being more than one ~ ~ l i l l i otilnes
n lower than their concentratio11 in
the cells. In case of cell destruction or injury by such cases as a damaged heart or
uncontrolled growth of cancer cells, the plasnla levels of these cellular enzynles are elevated
significantly. These changes in plasma concentration of particular enzymes are estimated by
the clinicians and used not only to detect cell damage but also to suggest the site of cell
damage. The degree of elevation of p1as111a concentrations of these enzyn~esalso gives a clue
to the extent of cellular damage. These enzyme assays have become a critical diagnostic tool
in the detection of heart, liver, pancreas, skeletal muscles, bone and malignant diseases. In
Table 6.4 you will find a list of sonle enzymes, clinical assay of which are used for detecting
particular diseased states.
Table 6.4 : Enzymes assayed in medical diagnostics
Enzyme Used in determination o f
Lactate dehydrogenase(LDH) heart or skeletal muscle damage
Alkaline phosphatase liver and bone disease
Serum glutamate oxaloacetate heart and liver disease
transaminase (SGOT)
Creatine phosphokipase (CK) myocardial infarction and muscle diseases
Acid phosphatase cancer of the prostate
a-Amylase panaeatitis

We shall illustrate the use of enzyme assay in myocardial infarction, which in simple
language means a heart attack. In this clinical condition the blood supply to the heart ~ ~ ~ u s c l e
is blocked and some of the cells of the heart nluscle die, thereby, releasing an abnormal
ainount of their enzymes into the blood. Monitoring the blood concentration of three enzymes
- CK, SGOT and LDH, helps a physician in identifying a heart attack and its severity. You
will find a graph of the concentration of these enzy~nesafter a heart attack in Fig. 6.20.

Fig. 6.20 : After a heart attack, concentration of CK increases rapidly and then falls. SGOT level
also increases rapidly and then falls. One to two days after an attack, LDH concentration
begins to increase, gradually rises for three to four days, and then decreases gradually
over the next seven to ten days.

Enzymes also find extensive use as laboratory reagents. A c o ~ n m o ntest, involving use of
enzymes, is to measure glucose concentration in urine, as in case of diabetes. Two enzymes,
n a ~ ~ l eglucose
ly oxidase and peroxidase, are used in this test. They are contained on a test
strip which is momentarily dipped in the urine sample. A colour change, on the strip after
sometime, is compared to a colour scale to measure the concentration of glucose. In this test
glucose oxidase catalyses conversion of glucose to gluconic acid and hydrogen peroxide.
Peroxidase then catalyses the reaction of hydrogen peroxide and o-toluidine to give a
coloured product, the colour intensity of which indicates the glucose concentration.
D - Glucose + 0, + H,O -
glucose oxidase
gluconic acid + H202,

peroxidase
o - toluidine + H202 -----+ coloured product + H 2 0
(scbiff base)
Enzymes are also used in treatment of niany medical problems. For example, trypsin and
chynlotrypsin are used in severe burns, for thev deconipose the proteins of the clotted blood,
pus and dead skin on the burned areas. Similarly many nasal sprays contain enzynles to clear
up congestion. And probably you might have used some digestive enzymes when you had a
heavy spicy meal, just to aid your digesl~on.

6.9 S U M M A R Y
Enzymes are biocatalysts which increase reaction rates enormously. They are
respol~siblefor no st of the che~nicalreactions which take place in the living
organis~l~s.
Enzynies are protein in nature, with considerable diversity of structure. Some enzymes
are simple proteins, whereas nlany arc conjugated proteins.
Many enzymes (conjugated proteins) require a nonprotein part without which they
cannot function. These no~lproteinparts, called cofactors, are either metal ions or low
molecular weight organic compouuds. These organic cofactors are mostly derived
from B group of vitamins. The organic cofactors are generally known as coenzymes.
Enzymes are characterised by an active site, where the substrate (reactant) binds,
uildergoes tra~lsfonnatio~i'and the11 the products are subsequently released. The active
site consists of amino acid side chains, some of which act as binding groups while
others act as catalytic groups. The a~llinoacid side chains of catalytic groups, are of
polar type only.
Enzymes are characterised by high catalytic efficiency, remarkable specificity and
regulatory properties. Enzymes can increase reaction rates a billion fold, which
enables biochenlical reactions to occur under physiological conditions. They are
highly specific, both with regard to the substrate and the reaction they catalyse. The
degree of specificity varies frtirn one enzyme to another. The regulatory properties of
enzymes enables fine tuning of enzyme activity in response to the needs of the cell,
under changing physiological conditio~ls.
Enzymes have been grouped into six major classes on the basis of the nature of the
reaction they catalyse. These classes are: Oxidoreductases, transferases, hydrolases,
lyases, isomerases and ligases. Each enzyme has been assigned a four number
identification code, indicating the major class, type of substrate or the bond cleaved,
the actual reaction catalysed and the serial number of the enzyme in its subsubclass.
Enzymes increase reaction rate enormously by lowering the energy of activation of the
reaction. This is achieved by bringing the reactants in close proximity a ~ l din a correct
orie~ltationfor the reaction to occur. Also at the active site, substrate is subjected to
stress and stmin, which facilitates bond making or breaking. The energy for this is
derived from favourable enzyme - substrate interactions at the active site.
Various factors govern the rate of enzymic reactions. The plot of enzyme velocity
versus substrate concentration shows a plateau at high substrate concentdtion,
indicating that the reaction rate after reaching a maximum value is independent of
further increase in substrate concentration.
The substrate concentration required for half maximal velocity (Vmax) is called the
Michaelis Constant Km.This is a useful parameter for determining catalytic potential
of an enzyme under physiological conditions and also gives an idea of the affinity of
the ellzylne for its substrate.
Enzyme activity is proportional to the co~lcentrationof the enzyme and is dependent
on pH and temperature of the mnedium.
Enzymes are inhibited by reagents which compete with the substrate for the active site.
These substances are known as competitive inhibitors. The other type of substances
which inhibit the enzyme by inactivating it are called~~~oncompetitive
inhibitors.
 -
BioardcaPla 11 Enzymes are regulated by the end product of a biosynthetic pathway. The final end
product inhibits the first enzyme of that pathway by binding to an allostenc site which
is distinct from the active site. The inhibition results from a conformational change in
the oligomeric structure of the first enzyme.
In another type of regulation, the amino acid side chains of enzymes are reversibly
modified, mainly by p$osphorylation or dephosphorylation. This produces an active or
an inactive enzyme.
Sometimes enzymes catalysing the same reaction exist in different electrophoretic
forms in different tissues of the same species. These different forms are know11 as
isoenzymes and result due to genetic factom. Lactate dehydrogenase is an interesting
case of isoenzymes.
Enzymes have a diagnostic value in medical sciences. Various enzyme assays are
employed to confirm, locate and also indicate the seventy of a disease in a human
being. Enzymes are used as laboratory reagents and are also employed in treating
various diseased conditions.

6.10 TERMINAL QUESTIONS

1. How does an oxidoreducbse differ from a transferase? Illustrate your answer with an
example.
2. How does the principle of competitive inhibition of enzyme action help in designing
drugs against bacterial diseases?
3. Describe active site of an enzyme.
4. Discuss the need for regulation of enzyme action.
5. Briefly describe important features of allosteric regulation.
6. Explain what is 'transition state' of a reaction?
7. How does an enzyme contribute to the lowering of energy of activation of a chemical
reaction?
8. Describe the origin of isoenzymes of lactage dehydrogenase.
9. What is K,,, and what does a low value ofK, signify?

6.11 ANSWERS
Self Assessment Questions

1. b and c
2. a and d
3. a - increase; b - lower, c - proximity and orientation; d - transition
4. d
5. c
-
6. a True; b - True; c - False; d - False
7. c
Terminal Questions

1. An oxidoreductase takes part in physiological oxidation-reductionprocess. On the other

hand, a transferase catalyses the transfer of a chemical group, from one substrate to
another. Lactate dehydrogenase is an example of an oxidoreductase, and hexokinase is
an example of a transferase.
2. Disease causing bacteria sometimes depend upon a particular enzyme reaction for their
survival and growth. Such bacteria can be killed by inhibiting the crucial enzyme
reaction by a con~petitiveinhibitor, which can be so designed as to partially resemble
the substrate. This coinpetitive inhibitor ordrug, displaces the real substrate from the
active site aiid inhibits eluylne activity, killing the bacteria.
 Enzymes
3. The portion of an enzyme which binds to the substrate aiid catalyses its chei~iical
transformation is called the active site of an enzyme. The active site has hydrophobic
and hydrophilic amino acids which bind to the substrate by hydrophobic, electrostatic
and hydrogen bonded interactions. Some of the hydrophilic amino acids, which are
charged or uncharged also participate in bond making and bond breaking processes.
4. Metabolic activities of the cell lead to the biosynthesis of cellular coinpoi~ei~ts
and to the
generation of energy needed to sustain life. Such a complex system of biochemical
transformations has to be regulated to channelise crucial metabolities into the required
pathway and not allow them to drift into the ultimate products of aerobic metabolisin
i.e., carbon dioxide and water. Since metabolism is driven by enzymes, it stailds to
reason that regulation of inetabolism is essentially regulation of enzyme action.
5. An important feature of allosteric regulation is that the end product of a pathway
inhibits the first enzyme of that pathway, by binding to an allosteric site ora site distinct
from the enzymic active site. The inhibition results from a conformational change in the
oligomeric structure of the first enzyme. Such allosteric enzymes do not show normal
kinetic behaviour, for in their case, the plot of rate versus concentration is not a
hyperbola but sigmoidal. The sigmoidal kinetics shows that interactioi~of the substrate
at one active site affects other active sites of the oligoineric structure through
inter-subunit contacts.
6. The energy profile of a chemical reaction shows that the reactants have to overcome an
energy barrier before they are converted into products. The structure and conformation
of reactant molecules corresponding to the peak position of this energy harrier is called
the 'transition state' of the reaction. This state represents molecules in their most
energised fonn where bonds are in the process of being made or broken.
7. Enzymes lower the energy of activation by bringing the reactants in close proximity,
and correctly orienting them towards each other, thus, in effect increasing their
concentration by several orders of magnitude. The entropic disadvantage which
chemical reactious suffer from, i.9 thus overcoine by the eiuyine. The energy for this
process is derived froin favourable enzyme- substrate interactioils at the active site. The
energy from these intelactiotls is also used to subject the substrate to stress and strain to
facilitate the bond making and bond breaking processes.
8. Lactate dehydrogenase isoenzymes consist of four subunits of two types, a and 0,
wbich are coded for by two different genes. These two types of subunits combine in
different proportions viz., ad, a3 f3 ,at p2, a p3 and p4, to give the five isoenzymes.
9. Concentration of the substrate expressed in moles per litre that gives half maximal
velocity of the enzyme, is known as K,. A small value of K, indicates high affinity of
UNIT 7 VITAMINS, COENZYMES AND
MINERALS

Structure
7.1 Introduction
Objectives

7.2 Biological Significance and Classification of Vitainills

7.3 Water Soluble Vitamins
The B Vitamins

Vitamin C

7.4 Fat Soluble Vitamins

7.5 Minerals and Trace Elements
Macrominerals

Trace Elemen&

7.6 Summary
7.7 Tenninal Questions
7.8 Answers

7.1 INTRODUCTION
You have already learnt from the preceding units that proteins, carbohydrates, fats and
nucleic acids are the inajor constituents of living cells, In Unit 6 we described a
specialised class of protein, the enzymes. You are aware by now that enzymes function as
hiological catalysts and through enzyme regulation, metabolism in the living organism is
also regulated. Life, as we inentioiled earlier, would not be possible without the
invaluable role of these molecules. However, you will recall that many enzyme
molecules require cofactors for their functioning. These cofactors can be small organic
~nolecules(coenzymes) or simple metal ions. In this unit you will lean1 about vitainills
and minerals, as many of these molecules perfonn the role of cofactors. However, the
role of vitamills and minerals, in general, is not only limited to being cofactors of
enzymes. It is only a part of their job. The cell, for its proper fu~lctioningalso requires
vitan~i~lsand soine other eleme~~ts, in trace amounts. These inolecules are essential for
nonnal growth, well being and proper fuilctioi~ii~g of human beings. In this unit we shall
briefly describe the vitamills, their deficiency symptoms, and dietary sources, their
relationship with coenzymes and their biochemical functions. We shall also discuss the
role of minerals and trace elements in the physiology and biochenlistry of humans. With
the knowledge gained from the study of various biomolecules, especially carbohydrates,
lipids and proteins, it will oe easier for you to learn about bioenergetics and n~etabolisnl.
This we shall describe in Block 3, where you will learn how the body converts food into
energy. You will be able to relate, more clearly, the role of enzymes, coenzynles and
other trace substances in metabolism, after studying Block 3.

Objectives
After studying this unit, you should be able to:
define vitamins and explain their biologi&l significance,
classify vitainills into water soluble and fat soluble types;
descrihe the biochemical functions of water soluble vitamins and also their role as
coenzymes in intermediary metabolism,
describe the mle of fat soluble vitatnitls, and
explain the significance of minerals and trace elements in human beings.