The study of the effects of such highly specific enzymes can give useful information

about the arrangement of amino acid residues in a protein.

Similarly, invertase is a hydrolyzing enzyme which hydrolyses the several fructosides,

viz. sucrose and methyl fructoside.

7.7. Mechanism of enzyme Activity

According to most acceptable hypothesis the enzyme combines with the substrate to
form an intermediate, enzyme-substrate complex, which thenbreaks down into product
[P] and enzyme back. The latter enzyme can againcombine with the fresh molecule of
the substrate in a similar manner. The formation of enzyme substrate complex as an
intermediate during the reaction has been proved by spectroscopic studies.

E + S  ES  E + P

In the formation of enzyme-substrate complexes, the substrate molecules attach at

certain specific points on the enzyme molecule. These specific point on enzyme
molecules where the substrate molecules attach are known as active, substrate or catalytic
sites. Active sites on the enzymes are usually provided be free hydroxyl group of serine
(as in trypsin, chymotrypsin, thrombin, alkaline phosphatase, cholinesterase and
elastase), phenolic group of tyrosine (as in pepsin), sulphurdyl group of cysteine, or
imidazole group of histidine.

7.7.1 Fischer's Lock and Key hypothesis

Initially, Fischer in 1984 proposed that the substrate fits into the active centre of the
enzymes as a key fits into the lock. This lock and key theory for enzyme action may be
shown diagramatically in Fig. 7.2
 Fig. 7.2

Thus according to the lock-and-key theory there are exact functional groups and
structural features in the enzyme into which the substrate molecule must fit. The region
of the enzyme that complexes with the substrate; called the active site. The theory is
somewhat restrictive in that it allows very little variation in substrate dimensions. These
are instances where although a substrate may complex through the lock-and-key
mechanism with an enzyme, no reaction ensures. Moreover, in certain cases a catalytic
activity is observed even though a 'fit' is impossible.

7.7.2 Koshland's Induced fit Hypothesis

In order to account for the above observations, the Fischer's lock-key mechanism was
modified by Koshland in 1963, in the form of induced fit mechanism. The essential
feature of the Koshland's induced fit model is the flexibility of the region of the active
site. In order words, we can say that in the Fischer model the active site is presumed to
be pre-shaped to fit the substrate, while in the induced fit model the substrate induces a
conformational change in the enzyme so that the substrate fits the active site in the most
convenient way. This explains why the enzymes become inactive in denaturation because
the latter phenomenon destroys the tertiary andquaternary structure of the enzyme and
thus the substrate can't induce conformation changes in the enzyme to fit the substrate
on the active sites.

Fig. 7.3: Representation of an induced fit by a conformational change in the enzyme

structure.
The active sites on the enzyme molecule exert a binding force on the substrate molecule
by hydrophilic as well as hydrophobic groups. Enzyme- substrate complexes are formed
by multiple bonding (covalent, hydrogen or electrostatic) with the substrate.

The functional groups of the active sites are arranged, in a definite spatial manner and
thus only those compounds which can fit into this definite spatial manner very well can
function as the substrate of the enzyme. This explains why only the d-isomer of a
single substance can actas substrate for a particular enzyme.

The enzymes requiring coenzymes for their activity also possess site for attachment of
coenzyme. The complex formed in such cases are known as enzyme-substrate-
coenzyme complexes.

7.7.3 Identification of Active Sites: Use of Inhibitors

Certain enzyme inhibitors particularly the competitive type function as drugs, hence
they are used to explain the action of drugs and in ascertaining the active sites on the
enzyme molecules. The first notable example where the sulphonamides which are
found to be the competitive inhibitors of p-amino benzoic acid (a bacterial growth
factor). p-Aminobenzoic acid forms a part of an essential coenzyme, called tetrafolic
acid, made by the bacterial cell.

The sulphonamide, being similar in structure, competitively inhibit the utilization of the
p-amino benzoic acid by the microorganism with the result folic acid production in the
micro-organism is prevented and thus growth of the micro-organism stops. It is
important to note that as the host obtains its folic acid requirement from the diet and does
not a synthesize it from the p- amino benzoic acid, it remains unaffected as far as folic
acid requirement is concerned. Hence sulphonamides can be toxic to micro-organisms
but virtually harmless to the host. Competitive inhibition also explains the action of
several of the drugs. For example, ephedrine and amphetamine prolong effects of the
hormones like adrenaline, noradrenaline and 5-hydroxytryptamine by competitively
inhibiting the enzyme monoamine oxidase (MAO) which brings about the oxidative
deamination of the above mentioned hormones.

A similar explanation has been put forward for the action of cocaine and of ant
depressive drugs like iproniazid and tranylcypromine.

The ability of some antidepressive drugs to inhibit monoamine oxidase may also have
serious side effects. Monoamine oxidase normally brings about the oxidation of tyramine
(formed by the decarboxylation of tyrosine), but in presence of MAO inhibitors this may
not happen and tyramine may thus enter the general circulation and releases noradrenalin
from the stores. The inhibitor also prolongs the action of noradrenalin andthe end-
result may be a very serious rise in blood pressure.

Another example of the drug whose function depends upon its competitive inhibiting
nature is allopurinol. The latter is very similar in structure to hypoxanthine and thus
inhibits the enzyme xanthine oxidase in bringing about the oxidation of hypoxanthine
and xanthine to uric acid and is thus used in the treatment of gout a disease in which
excess of uric acid is deposited.

7.8 ENZYME KINETICS

As has been pointed out rate of enzyme reaction depends on various factors, mainly
temperature, pH and substrate concentration. Kinetic studies by Michaelis-Menten and
Lineweaver-Burk, involving double reciprocal plot indicate formation of enzyme-
substrate complex.

7.8.1 MICHAELIS-MENTEN PLOT

It was Michaelis and Menten in 1913 who proposed a successful explanation for this
observation. According to them the enzyme and the substrate S combine rapidly to form
a complex, the enzyme-substrate complex, ES. This complex then breaks down
relatively slowly to form the product P of the reaction.

Applying law of mass action to the first step of the reaction. The rate of
forward reaction = K1 [E] [S]
The rate of backward reaction = K2 [ES]

Since at equilibrium the rates of the two reactions are equal,

K1[E][S] = K2 [ES] ................................................................... (1)

Divide both the sides by K1 [ES]

Now according to Michaelis and Menten, the slowest (rate determining) step of the,
reaction is the breakdown of the enzyme substrate complex, ES. Thus the velocity v of
the overall reaction, according to Lawof Mass action, is given by:

v = K3[ES]

Substitute the value of [ES] from equation (3).

Now if the enzymes are completely saturated with the substrate, v will increase to the
maximum velocity V which according to Law of Mass action will be given by

V = K3 [Eo]

Substitute the value of K3 [Eo] in equation (4)

This is the Michaelis-Menten equation. Now when v is equal to half of the maximum
velocity (V), i.e.

Thus the substrate concentration, [S], required for half Concentration of substrate [S] 

Fig. 7.5: Michaelis plot showing determination of Michaelis constant, Km

Attainment of the maximum activity of an enzyme is known as Michaelis constant. Thus

Michaelis constant may be determined from a plot, commonly known as Michaelis plot,
obtained by plotting substrate concentration, [S] versus rate of reaction, v (Fig. 7.5).
Michaelis constant is a characteristic of an enzyme, at definite pH and temperature.
Typically the values of Km lie in the range 10-2 to 10-5 moles/litre for various enzymes.
Km value is very useful in evaluating affinity of the enzyme for a substance; the low Km
value of an enzyme indicates its high affinity and vice versa. The Km value also gives an
idea regarding the type of inhibition of an enzyme caused by an inhibitor.

7.8.2 Line Weaver Plot

The value of Km may be obtained more accurately from the Lineweaver-Burk equation
which is obtained by inverting the Michaelis- Menton equation, (5). ,

This is known as Lineweaver-Burk equation. It is used for determining the Michaelis

constant for which the reciprocals of the observed velocities (I/v) are plotted against
the reciprocals of the correspondingsubstrate concentrations (1/S) keeping, the amount
constant (Fig. 7.6).

Fig. 7.6: Lineweaver-Burk plot to determine Km

A linear curve is obtained which when extrapolated intercepts the I/S line which gives
the value of -1/Km from which Km can easily be calculated.

Actually, the substrate binds to the active site of the enzyme molecule and as the
substrate is added more active sites of the enzyme molecule take part with the result the
reaction velocity increases. But since the number of active sites on an enzyme molecule
are fixed a stage will come when whole of them have combined with the substrate
molecules (saturation of the enzyme) at which stage the enzyme will be working at its
maximum velocity. Now since none of the active sites of the enzyme is free, further
addition of the substrate molecule will not further increase reaction velocity.

Fig. 7.7: Graph of reaction velocity against increasing

substrateconcentration at constant enzyme concentration.

The velocity of a catalysed reaction is proportional to the concentration of catalyst. In

case the enzyme concentration [E], is doubled, then as much as twice active sites become
available to combine with substrate provided an excess of substrate is present, and thus
the maximum velocity is also doubled. Therefore, in general v  [E]. This relationship
is illustrated in Fig. 7.8.

Fig. 7.8: Reversible and Irreversible Inhibition

7.8.3 Reversible and Irreversible Inhibition

Since enzymes are proteins, any agent which denatures proteins will inactivate enzymes.
Such agents are known as inhibitors of enzymes and thus may be defined as the chemical
substances that reduce the activity (i.e. velocity) of particular enzyme. They may be
small inorganic ions such as cyanide, which inhibits the enzyme called cytochrome
oxidase, or much more complex inorganic or organic molecules. This phenomenon in
which the enzyme activity is decreased by the presence of inhibitors is known as
inhibition. It may be of two types:

(a) Reversible Inhibition (b) Irreversible Inhibition

Reversible Inhibition

In this type of inhibition there is non-covalent bonding between inhibitor and enzyme.
In this type of inhibition there are many modes. These modes depend on the fact that how
and by what mechanism the mixing of inhibitor decreases the activity of enzyme or how
it affects kinetics of the reaction.

Reversible inhibitors easily associate and dissociate with the enzyme.

When they are bound with the enzyme, they make them active:

where I = Inhibitor.

Like ES complex, in EI complex also E is linked with I, using weak non-covalent

interaction. Reversible inhibition can be divided in to:

1. Competitive inhibition: This type of inhibition occurs when the so-called

inhibitor competes with the proper substrate for binding at the active site of the
enzyme. In such type of inhibitions, both enzyme-substrate (ES), and enzyme-
inhibitor (El) complexes are formed during the reaction. The relative amounts
of the two complexes depend partly upon the affinity of the enzyme towards the
substrate and inhibitor and partly upon the relative concentration of substrate and
the inhibitor. Thus if the inhibitor is present insufficiently high concentration, it
can displace the substrate entirely and thus blocks the reaction. On the other
hand, the degree (i.e. percentage) of inhibition observed for a competitive
inhibitor at a particular concentration can be reduced by increasing the substrate
concentration (see fig. 7.9).

Fig. 7.9: Michaelis plot for a competitive inhibitor

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