E+S ES E+P

Enzyme
In general, enzyme is a biocatalyst, which can be defined as a substance that
increases the velocity or rate of a chemical reaction inside the human/animal
body without itself undergoing any change in the overall process.
Again, it may be defined as biocatalyst synthesized by living cells. They are
protein in nature, colloidal and thermolabile in character, and specific in their
action.
– Enzymes have a high degree of specificity for their substrates.
– Enzymes accelerate chemical reactions tremendously.
– Enzymes can function in aqueous solution under mild conditions, which
are unlike the conditions that are frequently needed in organic chemistry.
– Almost all Enzymes are proteins.
Properties of Enzyme
1. They are made up of protein
2. They are heat liable
3. They are highly specific for their substrate
4. They possess an active site at which interaction with substrate takes place
5. They remain unchanged after reaction
6. They have enormous power of catalysis
7. Function of enzymes takes place in chemical reaction
8. Enzymes accelerate the rate of reaction by-
a) Not altering the reaction equilibrium
b) Being required in a very small amount, and
c) Being not consumed in the reaction
Prosthetic Groups
• Some enzymes require cofactors or prosthetic groups for their function. These typically are small organic
molecules or metals that bind to the protein, and that enable the protein to carry out its function.
• An enzyme that lacks its cofactors or prosthetic groups is called an apo-enzyme. An enzyme that contains
its cofactors or prosthetic groups is called a holo-enzyme.
• Cofactors from vitamins are called coenzymes.
Enzyme Cofactors
Enzymes are Powerful and Highly Specific
Catalysts
Enzymes are powerful, because-
• Enzymes accelerate reactions by factors of as much as a
million or more.
• The hydration of carbon dioxide is catalysed by an
enzyme namely, carbonic anhydrase. The transfer of CO2
from the tissues into the blood and then to the alveolar air
would be less complete in the absence of this enzyme.
• In fact, carbonic anhydrase is one of the fastest enzymes
known. Each enzyme molecule can hydrate 106
molecules of CO2 per second.
• This catalysed reaction is 107 times as fast as the
uncatalyzed one.
ENZYME KINETICS
Studying enzyme kinetics enables us to know-
a)The precise scheduling of reactions in a cell is important to the cell and our understanding of
its workings.
b) Enzyme mechanisms, e.g., the number of kinetic steps and the detailed chemistry can be
learned (enzymology).
c) Understanding enzyme function leads to better drugs.
Michaelis-Menten Equation
In 1913, two biochemists Leonor Michaelis and Maud Menten proposed a general theory for enzyme action and
derived a mathematical equation to express the hyperbolic shape of the curve and to calculate rate constants. They
proposed that enzyme molecules, E, and substrate molecules, S, combine in a fast and reversible step to form an ES
complex.
K1 k3
E+S ES E+P
K2 k4

There are two possible fates of the ES complex-

1. It can revert to free enzyme and substrate, or
2. It can proceed by a reversible reaction to form free enzyme and product, P.

The terms k1, k2, k3, and k4 define rate constants for the individual steps.

Now, we can simplify these reactions by considering the rate of reaction at times close to zero (hence, V o), when
there is negligible product formation and thus no back reaction.
To simplify matters, Briggs and Haldane suggested the steady state assumption in 1924 (where the
concentration of intermediate ([ES]) stay the same even if the concentrations of starting materials are
changing). This steady state occurs when the rates of formation and breakdown of the ES complex is equal.
Now from equation (2) and (3) we get –
k1[E][S] = (k2 + k3)[ES]
 [E] = [E]T - [ES]………………………… ……...(7)
Solving equation 6 we get,
[E][S] = KM [ES]
Or, ([E]T - [ES]) [S] = KM [ES]; [from equation 7]
Or, [E]T [S] – [ES] [S] = KM [ES]
Or, KM [ES] + [ES] [S] = [E]T [S]
Or, [ES] (KM + [S]) = [E]T [S]
Now we can plot a graph showing the effect of substrate concentration on reaction velocity for an enzyme-catalyzed
reaction.

From the graph we can see that-

1. at high concentrations of substrate ([S]>>K M), the velocity
of the reaction is zero order- that is, it is constant and
independent of substrate concentration.
2. At low concentrations of substrate ([S]<<KM), the velocity
of the reaction is first order- that is, it is proportional to
substrate concentration.

Fig. Michaelis-Menten kinetics

•Characteristics of Enzyme Reactions
1. Since, the enzyme is the catalyst, the higher the concentration, the greater should be the initial
reaction rate.
2. The reactions are most effective in optimal pH range of the enzymes.
3. Enzymes are also very sensitive to temperature changes.
- The rate for an enzyme-catalyzed reaction increases proportionally with increasing
temperature.
- For most enzymes, the rate decline begins in the temperature range of 50-60 oC.
4. The presence of metal ions and organic cofactors also influences the action of some enzymes.
5. Although many enzymes display the characteristics of Michaelis-Menten kinetics, some do not.
6. Enzyme action is generally reversible.
Enzyme Inhibition
Enzyme inhibitor is defined as a substance, which binds with the enzyme and brings
about a decrease in catalytic activity of that enzyme. There are two broad categories of
enzyme inhibition-
1. Reversible inhibition
2. Irreversible inhibition
Besides these, there is another broad category of inhibition, i.e., allosteric inhibition.

Reversible Inhibition
Here the inhibitor binds non-covalently with enzyme and the enzyme inhibition can be
reversed if the inhibitor is removed. The reversible inhibition is further subdivided into-
a. Competitive inhibition
b. Non-competitive inhibition
c. Un-competitive inhibition
Competitive Inhibition
The inhibitor (I) which is closely similar to the real substrate (S) and binds to the active site of the enzyme, is known
as competitive inhibitor.
i. The inhibitor competes with substrate and binds at the active site of the enzyme.
ii. When the inhibitor is bound, substrate is unable to bind, and vice versa.
iii. During the reaction, ES and EI complexes are formed as shown below-

iv. The quantitative extent of inhibition depends on the concentration ratio of substrate to inhibitor and the binding
affinity of each for the enzyme.
v. A very high concentration of substrate lessens the effect of the competitive inhibitor, unless the inhibitor has a much
higher binding affinity for the enzyme than does the substrate.
vi. The inhibitor molecule cannot be converted to a product, because it does not have the functional groups.
Example- The inhibition of the citric acid cycle enzyme, succinate dehydrogenase, by malonate, oxalate and
pyrophosphate provides a clear example of the competitive inhibition process.
Non-competitive inhibition (Mixed)
In non-competitive inhibition, both inhibitor and substrate can bind simultaneously to the enzyme molecule.
i. The two molecules must bind to different sites on the enzyme.
ii. The presence of inhibitor does not affect substrate binding but does interfere with the catalytic functioning of the
enzyme.
iii. The kinetic scheme for this inhibition is as follows.

Example- the reaction of a sulfhydryl group of a cysteine residue with a metal ion such as Ag +, Hg+, or Pb+2
represented by Mn+. here the sulfhydryl group is essential for catalytic action but does not participate in in substrate
binding.
Uncompetitive inhibition
An uncompetitive inhibitor is similar to noncompetitive inhibitor-it allows substrate to bind to the active site, but the
inhibitor binds only to the ES complex.
- It will influence the activity of the enzyme only when substrate concentrations, in turn, ES concentrations are
high.
- The kinetic scheme for this inhibition is as follows.
Irreversible Inhibition
The inhibitors bind with the enzymes and inactive them, which is irreversible. These inhibitors are usually toxic
substances.
E---H- active enzyme, R—X- irreversible inhibitor, E—R- modified enzyme, i.e., catalytically inactive.

E------H + R------X E-----R + HX

Allosteric Inhibition
Some of the enzymes possess additional sites besides the active site. Such enzymes are known as allosteric enzyme.
The enzyme activity is increased when a positive allosteric effector binds at the activator site; but when a negative allosteric
effector binds at the activator site that inhibits the enzyme activity.