FACTORS AFFECTING ENZYME ACTIVITY

The contact between the enzyme and substrate is the most essential pre-requisite for
enzyme activity. The important factors that influence the velocity of the enzyme reaction are
discussed hereunder
1. Concentration of enzyme
As the concentration of the enzyme is increased, the velocity of the reaction proportionately
increases. In fact, this property of enzyme is made use in determining the serum enzymes for the
diagnosis of diseases.
By using a known volume of serum, and keeping all the other factors (substrate, pH,
temperature etc.) at the optimum level, the enzyme could be assayed in the laboratory.

2. Concentration of substrate
Increase in the substrate concentration gradually increases the velocity of enzyme
reaction within the limited range of substrate levels. A rectangular hyperbola is obtained when
velocity is plotted against the substrate concentration.
 Three distinct phases of the reaction are observed in the graph (A-linear; B-curve; C-
almost unchanged).
Order of reaction: When the velocity of the reaction is almost proportional to the substrate
concentration (i.e. [S] is less than Km), the rate of the reaction is said to be first order with respect
to substrate. When the [S] is much greater than Km, the rate of reaction is independent of substrate
concentration, and the reaction is said to be zero order.

Enzyme kinetics and Km value

The enzyme (E) and substrate (S) combine with each other to form an unstable enzyme
substrate complex (ES) for the formation of product (P).

Here k1, k2 and k3 represent the velocity constants for the respective reactions, as indicated
by arrows.
Km, the Michaelis-Menten constant (or Brig’s and Haldane’s constant), is given by the formula

The following equation is obtained after suitable algebraic manipulation.

Let us assume that the measured velocity (v) is equal to .Then the equation (1) may be
substituted as follows

K stands for a constant and m stands for Michaelis (in Km).

Km or the Michaelis-Menten constant is defined as the substrate concentration (expressed in
moles/l) to produce half-maximum velocity in an enzyme catalysed reaction. It indicates that
half of the enzyme molecules (i.e. 50%) are bound with the substrate molecules when the substrate
concentration equals the Km value.
Km value is a constant and a characteristic feature of a given enzyme (comparable to a
thumb impression or signature). It is a representative for measuring the strength of ES complex. A
low Km value indicates a strong affinity between enzyme and substrate, whereas a high Km
value reflects a weak affinity between them. For majority of enzymes, the Km values are in the
range of 10–5 to 10–2 moles. It may however, be noted that Km is not dependent on the
concentration of enzyme.
Lineweaver-Burk double reciprocal plot:
For the determination of Km value, the substrate saturation curve is not very accurate since
Vmax is approached asymptotically. By taking the reciprocals of the equation (1), a straight line
graphic representation is obtained.

The above equation is similar to y = ax + b. Therefore, a plot of the reciprocal of the

velocity (1/v) vs. the reciprocal of the substrate concentration (1/[S]) gives a straight line. Here the
slope is Km/Vmax and whose y intercept is 1/Vmax. The Lineweaver-Burk plot is shown in below.

It is much easier to calculate the Km from the intercept on x-axis which is –(1/Km).
Further, the double reciprocal plot is useful in understanding the effect of various inhibitions.

3. Effect of temperature
Velocity of an enzyme reaction increases with increase in temperature up to a maximum
and then declines. A bell-shaped curve is usually observed.
Temperature coefficient or Q10 is defined as increase in enzyme velocity when the temperature
is increased by 10°C. For a majority of enzymes, Q10 is 2 between 0°C and 40°C. Increase in
temperature results in higher activation energy of the molecules and more molecular (enzyme and
substrate) collision and interaction for the reaction to proceed faster.
The optimum temperature for most of the enzymes is between 35°C–40°C. However, a few
enzymes (e.g. Taq DNA polymerase, muscle adenylate kinase) are active even at 100°C. Some
plant enzymes like urease have optimum activity around 60°C. This may be due to very stable
structure and conformation of these enzymes.
In general, when the enzymes are exposed to a temperature above 50°C, denaturation
leading to derangement in the native (tertiary) structure of the protein and active site are seen.
Majority of the enzymes become inactive at higher temperature (above 70°C).

Clinical significance: Foods can be preserved in refrigerators (at low temperatures) due to
reduced bacterial enzyme activities. Certain surgeries are carried out by lowering the patient’s
body temperature (induced hyperthermia), and thus the metabolic rate.
4. Effect of pH
Increase in the hydrogen ion concentration (pH) considerably influences the enzyme
activityand a bell-shaped curve is normally obtained.
 Each enzyme has an optimum pH at which the velocity is maximum. Below and above this
pH, the enzyme activity is much lower and at extreme pH, the enzyme becomes totally inactive.
Most of the enzymes of higher organisms show optimum activity around neutral pH (6-8).
There are, however, many exceptions like pepsin (1-2), acid phosphatase (4-5) and alkaline
phosphatase (10-11). Enzymes from fungi and plants are most active in acidic pH (4-6). Hydrogen
ions influence the enzyme activity by altering the ionic charges on the amino acids (particularly at
the active site), substrate, ES complex etc.
5. Effect of product concentration
The accumulation of reaction products generally decreases the enzyme velocity. For certain
enzymes, the products combine with the active site of enzyme and form a loose complex and, thus,
inhibit the enzyme activity. In the living system, this type of inhibition is generally prevented by
a quick removal of products formed.
6. Effect of activators
Some of the enzymes require certain inorganic metallic cations like Mg2+, Mn2+, Zn2+,
Ca2+, Co2+, Cu2+, Na+, K+ etc. for their optimum activity. Rarely, anions are also needed for enzyme
activity e.g. chloride ion (Cl–) for amylase. Metals function as activators of enzyme velocity
through various mechanisms— combining with the substrate, formation of ES-metal complex,
direct participation in the reaction and bringing a conformational change in the enzyme.
Two categories of enzymes requiring metals for their activity are distinguished
  Metal-activated enzymes : The metal is not tightly held by the enzyme and can be
exchanged easily with other ions
e.g. ATPase (Mg2+ and Ca2+),
Enolase (Mg2+)
 Metalloenzymes : These enzymes hold the metals rather tightly which are not readily
exchanged. e.g. alcohol dehydrogenase, carbonic anhydrase, alkaline phosphatase,
carboxypeptidase and aldolase contain zinc.
Phenol oxidase (copper);
Pyruvate oxidase (manganese);
Xanthine oxidase (molybdenum);
Cytochrome oxidase (iron and copper).
7. Effect of time
Under ideal and optimal conditions (like pH, temperature etc.), the time required for an
enzyme reaction is less. Variations in the time of the reaction are generally related to the alterations
in pH and temperature.
8. Effect of light and radiation
Exposure of enzymes to ultraviolet, beta, gamma and X-rays inactivates certain enzymes
due to the formation of peroxides. e.g. UV rays inhibit salivary amylase activity.

ENZYME ACTION

ACTIVE SITE
Enzymes are big in size compared to substrates which are relatively smaller. Evidently, a
small portion of the huge enzyme molecule is directly involved in the substrate binding and
catalysis.
The active site (or active centre) of an enzyme represents as the small region at which
the substrate(s) binds and participates in the catalysis.
MECHANISM OF ENZYME ACTION
Catalysis is the prime function of enzymes. Enzymes are powerful catalysts. The nature of
catalysis taking place in the biological system is similar to that of non-biological catalysis. For any
chemical reaction to occur, the reactants have to be in an activated state or transition state.

Enzymes lower activation energy: The energy required by the reactants to undergo the reaction
is known as activation energy. The reactants when heated attain the activation energy. The
catalyst (or the enzyme in the biological system) reduces the activation energy and this causes the
reaction to proceed at a lower temperature. Enzymes do not alter the equilibrium constants, they
only enhance the velocity of the reaction. The role of catalyst or enzyme is comparable with a
tunnel made in a mountain to reduce the barrier as illustrated below:
 The enzyme lowers energy barrier of reactants, thereby making the reaction go faster. The
enzymes reduce the activation energy of the reactants in such a way that all the biological systems
occur at body temperature (below 40°C).
Enzyme-substrate complex formation
The prime requisite for enzyme catalysis is that the substrate (S) must combine with the
enzyme (E) at the active site to form enzyme substrate complex (ES) which ultimately results in
the product formation (P).

A few theories have been put forth to explain mechanism of enzyme-substrate complex formation.
1. Lock and key model or Fischer’s template theory
This theory was proposed by a German biochemist, Emil Fischer. This is in fact the very
first model proposed to explain an enzyme catalysed reaction. According to this model, the
structure or conformation of the enzyme is rigid. The substrate fits to the binding site (now active
site) just as a key fits into the proper lock or a hand into the proper glove. Thus the active site of
an enzyme is a rigid and pre-shaped template where only a specific substrate can bind. This model
does not give any scope for the flexible nature of enzymes, hence the model totally fails to explain
many facts of enzymatic reactions.
 2. Induced fit theory or Koshland’s model
Koshland, in 1958, proposed a more acceptable and realistic model for enzyme substrate
complex formation. As per this model, the active site is not rigid and pre-shaped. The essential
features of the substrate binding site are present at the nascent active site. The interaction of the
substrate with the enzyme induces a fit or a conformation change in the enzyme, resulting in the
formation of a strong substrate binding site.
Further, due to induced fit, the appropriate amino acids of the enzyme are repositioned to
form the active site and bring about the catalysis. Induced fit model has sufficient experimental
evidence from the X-ray diffraction studies. Koshland’s model also explains the action of
allosteric modulators and competitive inhibition on enzymes.

3. Substrate strain theory

In this model, the substrate is strained due to the induced conformation change in the
enzyme. It is also possible that when a substrate binds to the preformed active site, the enzyme
induces a strain to the substrate. The strained substrate leads to the formation of product.
In fact, a combination of the induced fit model with the substrate strain is considered to be
operative in the enzymatic action.