ENZYMES

Introduction- The metabolic reactions that occur in cells

are so slow that they could not contribute to life unless the cells
did something to speed them up. That is the role of bio-
catalysts (enzymes): -substances that speed up a reaction
without being permanently altered by that reaction. A catalyst(in
a biological system- it’s enzyme) does not cause a reaction that
would not take place eventually without it, but merely speeds up
the rates of both forward and backward reactions, allowing
equilibrium to be approached faster. But Enzymes do more than
merely speed up metabolic processes. They also control them.
Enzymes ensure that metabolism proceeds by gentle steps in an
orderly fashion.

Definition: Enzymes are water soluble, colloidial organic

biomolecules which are wholly or partially proteinous in nature
and capable of catalyzing specific biochemical reaction under
specific conditions.

Properties of Enzymes:

1. Enzymes are polymeric biomolecules with higher

molecular wt.

2. Enzymes are wholly or partially proteinous (globular

protein) in nature
(except Ribozyme & RNAase-P two non-proteinous
enzymes in which RNA
acts as a catalysts. )

3. They are required in small amounts and effects the

change of a large
amount of substrate.

4. They are not altered irreversibly during the course of

the reaction, and
 therefore each enzyme molecule can participate
repeatedly in individual
reaction.

5. Enzymes usually have 3- dimensional tertiary structure

maintained by H-
bonds, ionic bond, disulphide bond and hydrophobic
forces.

6. They act as biocatalysts –help in reaching the

equilibrium constant faster
by lowering the required activation energy .

7. They have no effect on the thermodynamics of the

reactions-Enzymes do
not supply energy for a chemical reaction and therefore
do not determine
whether a reaction is thermodynamically favourable or
unfavourable.

8. Enzymes are substrate specific –e.g. an enzyme can

catalyse only a
single or a particular type of reaction.

9. Enzymes have high turnover number i.e. they generally

work very rapidly.

10. Enzymes activity varies with temperature, pH,

pressure and substrate
& enzyme concentrations.

Classification of Enzymes:

A. On the basis of site of activity-

i) Intracellular Enzyme: An enzyme that remains active

only within the
 cell in which it is formed.

ii) Extracellular Enzyme: An enzyme, such as a digestive enzyme,

that
functions outside the cell from which it originates.

B. On the basis of composition:

i) Simple Enzymes- Enzymes composed wholly of protein.

ii) Complex Enzymes- Enzymes, which are composed of protein plus a

relatively small organic molecule.

N.B- Complex enzymes are also known as holoenzymes. In

this terminology the protein component is known as the
apoenzyme, while the non-protein component is known as
the coenzyme or prosthetic group where prosthetic group
describes a complex in which the small organic molecule is
tightly bound
N.B.-Enzymes to the
that apoenzyme
require a metal (by covalent
in their bonds); are
composition whenknown
the binding between the apoenzyme and non-protein
as metalloenzymes if they bind and retain their metal atom(s)
components
under is in loosely
all conditions manner
with very (by non-covalent),
high affinity. Those whichthe small
have a
organic molecule is called a coenzyme. Many prosthetic
lower affinity for metal ion, but still require the metal ion for
groupsare
activity, andknown
coenzymes are water-soluble
as metal-activated derivatives of
enzymes.
vitamins.
C. On the basis of function:- Currently enzymes are grouped
into six functional classes by the International Union of
Biochemists (I.U.B.).

NUMBE CLASSIFICATIO
BIOCHEMICAL PROPERTIES
R N

Act on many chemical groupings to add or remove

1 Oxidoreductases
hydrogen atoms. (=catalyze a redox reaction.)
 Transfer functional groups between donor and
acceptor molecules. Kinases are specialized
2 Transferases transferases that regulate metabolism by
transferring phosphate from ATP to other
molecules.

3 Hydrolases Add water across a bond, hydrolyzing it.

Add water, ammonia or carbon dioxide across

double bonds, or remove these elements to
4 Lyases
produce double bonds. (=break C-O, C-C, or C-N
bonds).

Carry out many kinds of isomerization: L to D

5 Isomerases isomerizations, mutase reactions (shifts of
chemical groups) and others.

Catalyze reactions in which two chemical groups

6 Ligases are joined (or ligated) with the use of energy. from
ATP.

Enzyme Specificity: In general, there are four distinct

type of specificity-

1. Absolute specificity- the enzyme will catalyze only one

reaction.

2. Group specificity- the enzyme will act only on molecules that

have specific/
same functional groups, such as amino, methyl etc.
3. Linkage specificity- the enzyme will act on a particular type
of chemical bond
regardless of the rest of the molecular structure.

4. Stereochemical (optical) specificity- the enzyme will act on

a particular
steric or optical isomer.

Mechanism of Enzyme Action

How enzymes speed up reactions?
All chemical reactions have a potential energy barrier that must
be overcome to initiate the reactions. So, all reactant molecules
must possess sufficient energy to overcome the energy barrier/
enter the reactive state (=Transition state) that lies at the apex of
the energy barrier.(Fig.1-a,b,c).
 Fig.1-a,b,c
In living organisms, enzymes act as bio-catalyst and lower the
activation energy (Ea) of the reaction( Fig.-2). It is considered that
the enzymes combine with the substrate molecules and bring
them close together which favours their collisions in the most
suitable directions and locations for the reactions to occur.
 Fig.-2
A comparison between catalyzed and non-catalyzed reactions
shows (Fig.-2) that there is no difference in the energy of the
reactants and products. This confirmed that enzyme does not
change the thermodynamics of the system but changes the
pathway of reaching the final state(=providing alternative
pathway).
An enzyme combines with its substrate to form a short-lived
enzyme-substrate comlex. Due to the the formation of this
complex the chances of occurrence of reaction are greatly
enhanced. Once the reaction has started to its optimum level, the
enzyme-substrate complex first transforms into enzyme-product
complex and then breaks up into product/s and enzyme.
substrate + enzyme enzyme/substrate complex enzyme-
prooduct(s) complex enzyme + product(s)

Activation Energy- The minimal kinetic energy

required/needed for a reactant to undergo a chemical
reaction.
 Remember the point:
Enzyme lowers the entropy of the Substrate and thus facilitates
the reaction.

Models (Hypothesis) of Enzyme Action:

1. Lock & Key Hypothesis: It was put forward by Emil
Fisher in
1894. According to this hypothesis, both enzyme and
substrate
molecules have specific geometrical shapes. Enzyme
bears active
site in which specific type of substrate molecules can
form a
complex association by their specific complementary
structure,
called reactive site. This is referred to as the “Lock &
Key”
hypothesis, where the substrate is the key whose
shape is
is complementary to the key hole (active site) of
the lock
or enzyme .(fig.-3)
 Fig.-3
substrate + enzyme enzyme/substrate complex
enzyme-prooduct(s) complex enzyme
+ product(s)

(Source- i) Cell & Molecular Biology by Robertis &

Robertis,
ii) Biological Science by Green et. ( Cambridge),
iii) Text Book of Biochemistry, edited by T
Devlin.)

Remember the point-

This model explains enzyme specificity, but it fails to explain the

stabilization of the transition state that enzymes achieve.
2. Induced fit mechanism: In 1959 Khosland
suggested a modification
to the lock & key hypothesis and proposed induced fit
mechanism. According
to this Hypothesis the active site of the enzyme does not
initially exist in a
shape that is complementary to the substrate but is induced to
assume the
complementary shape in the presence of specific substrate. In
Khosland
language, “The active site is induced to assume a
complementary shape in much
of the same way as a hand induces a change in the shape of a
glove”. So,
according to this model, the structre of the enzyme (or its active
site) is flexible.
The active site of the enzyme contains two groups-a)
Buttressing group- it
supports the substrate. b) Catalytic group- it catalyze the the
reaction.
When substrate comes in contact with the the buttressing group,
the active
site changes to bring the catalytic group opposite the substrate
bonds to be
broken. (Fig.-4)
Fig.-4
 Allosteric Enzymes: In addition to simple enzymes that interact only
with substrates and inhibitors, there is a class of enzymes that bind small,
physiologically important molecules and modulate activity in ways other than those
described above. These are known as allosteric enzymes; the small regulatory
molecules to which they bind are known as effectors. Allosteric effectors bring about
catalytic modification by binding to the enzyme at distinct allosteric sites, well removed
from the catalytic site, and causing conformational changes that are transmitted through
the bulk of the protein to the catalytically active site(s).

The hallmark of effectors is that when they bind to enzymes, they alter the catalytic
properties of an enzyme's active site. Those that increase catalytic activity are known as
positive effectors. Effectors that reduce or inhibit catalytic activity are negative effectors.

Definition- Allosteric enzymes are those enzymes whose activities

are regulated by some non-substrate molecules that bind to the
enzymes well away from the active site.
Points to be remembered:
1. Allosteric enzymes are regulated by molecules called allosteric
modulators or effectors.
2.Allosteric modulators arte two types:
a) Allosteric activators =Speed up the reaction
b) Allosteric inhibitors =slow down the reaction.

3.Alloesteric modulators bind to the enzymes non-covently at the site

other than the active site.

4. Allosteric enzymes do not obey Michelis-menton or Km constant, instead

it gives sigmoid kinetics. ( Explained later)

5. Allosteric enzymes generally are a part of a metabolic pathway, known

as feedback mechanism.

6. Example-Enzyme phosphofructokinase is activated by ADP and inhibited

by ATP.
Factors affecting Enzyme activity:
1. Temperature: Enzymes are temp. specific as each
enzyme is most active
at a specific temp. called optimum temp. Range of optimum
temp. for most of
the enzymes is :25-400C.Any increase or decrease in temp
than the optimum
temp. decrease the enzyme activity.

In the above figure the temperature optima of three different enzymes is depicted. You
should note that the temperature optimum of each enzyme is different.

(The Curve in blue might represent an enzyme isolated from a shrimp that normally lives
in the cold waters of Alaska. Thus its enzymes have evolved to work best at lower temperatures.

The curve in red might represent that obtained with porcine chymotrypsin.

Curve curve in green might represent the temperature optimum obtained with an enzyme
isolated from a bacteria that normally lives in the hot springs of Yellowstone National Park. The
enzymes from this bacteria would work best at temperatures that would normally denature
enzymes isolated from you or me.
 In addition, you should notice that not only are the optimum temperatures different, the shapes
of the curves are also different)

Temperature Coefficient: The effect of the temperature on the rate of

a reaction can be expressed as the temperature coefficient,Q10

0
Q10 =rate of a reaction at (t+10) C rate of reaction at t0 C.

Over a range of 00 -400 C, Q10 , for enzyme controlled reaction is 2. In other words,
0
the rate of an enzyme-controlled reaction doubles with every 10 C rise in

temperature.

But high temp. causes sudden fall in enzyme activity because the secondary & tertiary

structure have been disrupted due to breaking of H-bonds & disulphide bonds. This is

known as denaturation of enzyme.

H: Under conditions of constant temperature, every enzyme functions most efficiently

2. P

at a particular hydrogen ion concentration called optimum PH .When the PH is altered above or below

the optimum value the rate of enzyme activity diminishes. Changes in PH alter the acidic or basic

groups that help to maintain the specific shape of the enzyme ie, PH change leads to an alteration in

enzyme shape particularly at its active site.

For example in the figure above is represented a situation in
which two different enzymes might have very different pH optima.
The one depicted by the green curve might represent the pH
optimum for the enzyme pepsin which degraded proteins
(protease) in the acidic lumen of the stomach. The second curve
(in red) might represent the enzyme carbonic anhydrase that
works in the neutral pH of our cytosol.

3. Concentration of enzyme and substrate: The rate of

an enzyme-catalysed reaction depends on the concentrations of
enzyme and substrate. As the concentration of either is increased
the rate of reaction increases

i)For a given enzyme conc. increasing the substrate

concentration increases the rate of reaction (enzyme activity).
However, enzyme saturation limits reaction rates. An enzyme is
saturated when the active sites of all the molecules are occupied.
At the saturation point, the reaction will not speed up, no matter
how much additional substrate is added. The graph of the
reaction rate will plateau."

ii) Provided that the substrate concentration is high and that

temperature and pH are kept constant, the rate of reaction is
proportional to the enzyme concentration.
 Enzyme Kinetics & Michelis-Menton Equation
Kinetics is the study of the rate of change of the initial state of reactant/s
and product/s
to the final stage of reactant/s and product/s.

The Michaelis-Menten equation is a quantitative description of the

relationship among the rate of an enzyme- catalyzed reaction [v1], the
concentration of substrate [S] and two constants, Vmax and Km (which are set
by the particular equation). The symbols used in the Michaelis-Menten
equation refer to the reaction rate [v1], maximum reaction rate (Vmax),
substrate concentration [S] and the Michaelis-Menten constant (Km).
According to this postulation the enzyme-substrate reaction proceeds in two
steps :
i) The first step can be represented as bellow:
K1
E+S ( ES) ......... (1)
K2

ii) In the 2nd steps (ES) complex breaks down to form the product and
free enzyme:

K3
(ES) E+P........ (2) (K1,K2,K3,K4 are rate constant for the
reactions) K4

As shown in Fig- below the velocity of the reaction depends

on the the substrate concentration(S). At low Conc. the initial velocity(V) of
the reaction describes a hyperbola. however, As(S) increases the reaction
saturates and a plateau. At this point , which corresponds to Vmax, all the
enzyme is in the form of (ES) complex. The equation of the the curve is :
Vmax(S)
V= ....... (3)
Km=istheMichaelisconstant,whichmaybedefined asthe (S)* atwhichhalf of
are forming (ES) complexes.
theenzymesmolecules

The smaller the value of Km, the greater the apparent affinity of the
enzyme for the substrate.

(S)*=substrateConc.
4. Presence of Inhibitors: Inhibitors are those chemical compounds
which decrease or stop the catalytic activity of the enzymes and the
phenomenon is known as inhibition. It is of following types:

Inhibitor Type Binding Site on Enzyme Kinetic effect

Specifically at the catalytic

site, where it competes with
Competitive substrate for binding in a
Inhibitor dynamic equilibrium- like
process. Inhibition is reversible
by substrate.
Vmax is unchanged; Km,
as defined by [S]
required for ½ maximal
activity, is increased.

Binds E or ES complex other

than at the catalytic site.
Noncompetitive Substrate binding unaltered,
Inhibitor but ESI complex cannot form
products. Inhibition cannot be
reversed by substrate.
Km appears unaltered;
Vmax is decreased
proportionately to
inhibitor concentration.

Binds only to ES complexes at

locations other than the
Apparent Vmax
catalytic site. Substrate
decreased; Km, as
Uncompetitive binding modifies enzyme
defined by [S] required
Inhibitor structure, making inhibitor-
for ½ maximal activity,
binding site available.
is decreased.
Inhibition cannot be reversed
by substrate.
NB- i) Reversible inhibitors can be divided into two main categories;
competitive inhibitors and noncompetitive inhibitors, with a third
category, uncompetitive inhibitors, rarely encountered.
ii) The hallmark of all the reversible inhibitors is that
when the inhibitor concentration drops, enzyme activity is
regenerated. Usually these inhibitors bind to enzymes by
non-covalent forces and the inhibitor maintains a
reversible equilibrium with the enzyme.