Factors affecting enzyme activity

Factors affecting enzyme
activity
Namrata Chhabra
M.D.Biochemistry
15-May-20 Namrata Chhabra 1

What are Enzymes ?
• Enzymes are biological catalysts of the chemical reactions.
• They are neither consumed nor permanently altered as a
consequence of their participation in a reaction.
• With the exception of catalytic RNA molecules, or ribozymes, all
enzymes are proteins in nature.

Enzyme catalyzed reaction
• The basic enzymatic reaction can be represented as follows:
• where E represents the enzyme catalyzing the reaction, S the
substrate, the substance being changed, and P the product of the
reaction.

Enzyme Catalysis- overview

Thermodynamic perspective of mechanism of
enzyme action
• All enzymes accelerate reaction
rates by providing transition
states with a lowered activation
energy for formation of the
transition states.
• The lower activation energy
means that more molecules
have the required energy to
reach the transition state.

Thermodynamic changes-overview

Enzyme kinetics
• The rate of a chemical reaction is described by the number of
molecules of reactant(s) that are converted into product(s) in a
specified time period.
• International unit – One IU is defined as the activity of the enzyme
which transforms one micro mole of substrate into products per
minute per litre of sample under optimal conditions and at defined
temperature . It is expressed as IU/L
• Katal – catalytic unit – One Katal is defined as the number of moles
of substrate transformed per second per litre of sample. It is
abbreviated as kat or k.

Numerous factors affect the reaction rate
Reaction rate
Substrate
concentration
Enzyme
concentration
Product
concentration
TemperaturepH
Time
Activators
and inhibitors

Effect of substrate concentration
• At lower concentrations, the active sites on most of the enzyme
molecules are not filled because there is not much substrate.
• Higher concentrations cause more collisions between the molecules.
• The rate of reaction increases(First order reaction).

Effect of substrate concentration (contd.)
• The maximum velocity of a reaction is reached when the active sites
are almost continuously filled.
• Increased substrate concentration after this point will not increase
the rate.
• Reaction rate therefore increases as substrate concentration is
increased but it levels off (Zero order reaction)

Effect of substrate concentration
The shape of the curve that
relates activity to substrate
concentration is hyperbolic. First order
reaction
Zero order reaction

• At points A or B, increasing
or decreasing [S] therefore
will increase or decrease the
number of ES complexes
with a corresponding change
in vi.
• The rate of reaction is
substrate dependent (First
order reaction).
• At point C essentially all the
enzyme is present as the ES
complex. Since no free
enzyme remains available for
forming ES, further increases
in [S] cannot increase the
rate of the reaction .
• Reaction rate therefore
becomes independent of
substrate concentration
(Zero order reaction).

km and its significance
• Michaelis constant, km is the substrate concentration at which V1 is
half the maximal velocity (Vmax /2) attainable at a particular
concentration of enzyme.
• km thus has the dimensions of substrate concentration.
• It is specific and constant for a given enzyme under defined
conditions of time , temperature and p H
• Km determines the affinity of an enzyme for its substrate, lesser the
Km for is the affinity and vice versa
• Km value helps in determining the true substrate for the enzyme.

Effect of enzyme concentration
As the amount of enzyme is increases
the rate of reaction increases. If there
are more enzyme molecules than are
needed, adding additional enzyme
will not increase the rate. Reaction
rate therefore increases as enzyme
concentration increases but then it
levels off.

Effect of product concentration
• The enzyme activity declines when the products start accumulating.
This is called product or feed back inhibition.
• Under certain conditions reverse reaction may be favored forming
back the substrate.

Effect of Temperature
• Raising the temperature increases the kinetic energy of molecules.
• Increasing the kinetic energy of molecules also increases their motion
and therefore the frequency with which they collide.
• This combination of more frequent and more highly energetic and
productive collisions increases the reaction rate.

Temperature coefficient
• The Q10, or temperature coefficient, is the factor by which the rate of
a biologic process increases for a 10 °C increase in temperature.
• For the temperatures over which enzymes are stable, the rates of
most biologic processes typically double for a 10 °C rise in
temperature (Q10 = 2). (A ten degree Centigrade rise in temperature
will increase the activity of most enzymes by 50 to 100%).

• The reaction rate increases with
temperature to a maximum level,
then abruptly declines with further
increase of temperature.
• Most animal enzymes rapidly
become denatured at temperatures
above 40oC.

• The optimal temperatures of the enzymes in higher organisms rarely
exceed 50 °C.
• Some enzymes lose their activity when frozen.
• Enzymes from thermophilic bacteria found in hot springs are active at
100 °C.
• Changes in the rates of enzyme-catalyzed reactions that accompany a
rise or fall in body temperature constitute a prominent survival
feature for "cold-blooded" life forms such as lizards or fish, whose
body temperatures are dictated by the external environment.

Effect of Hydrogen Ion Concentration (pH)
• The rate of almost all enzyme-
catalyzed reactions exhibits a
significant dependence on
hydrogen ion concentration.
• Most intracellular enzymes
exhibit optimal activity at pH
values between 5 and 9.
• When the activity is plotted
against pH, a bell-shaped curve is
usually obtained

Effect of
Hydrogen Ion
Concentration
(pH)
The relationship of activity to
hydrogen ion concentration
reflects the balance between
enzyme denaturation at high
or low pH
The binding and recognition of
substrate molecules with
dissociable groups of enzymes
typically involves the
formation of salt bridges.
The most common charged
groups are the negative
carboxylate groups and the
positively charged groups of
protonated amines.
Gain or loss of critical charged
groups thus will adversely
affect substrate binding and
thus will retard or abolish
catalysis.

Effect of Hydrogen Ion
Concentration (pH)
• The pH optimum—i. e., the pH value at which
enzyme activity is at its maximum—is often close
to the pH value of the cells (i. e., pH 7).
• There are also exceptions to this.
• The proteinase pepsin , which is active in the
acidic gastric lumen, has a pH optimum of 2, while
alkaline phosphatase is active at pH values higher
than 9

Effect of
activators and
Coenzymes
• The activity of certain enzymes is greatly
dependent on metal ion activators and
coenzymes.
• Vitamins act as coenzymes in a variety of
reactions.

Effect of modulators and Inhibitors
• The enzyme activity is reduced in the presence of an inhibitor and
• the enzyme activity may be increased in the presence of a positive
modifier.

Effect of Time
• The longer an enzyme is incubated with its substrate, the greater the
amount of product that will be formed.
• However, the rate of formation of product is not a simple linear
function of the time of incubation.
• All proteins suffer denaturation, and hence loss of catalytic activity,
with time.

Effect of Time
• Enzyme catalyzed
reactions undergo
completion in an
optimum time.
• Variations of time
affect the rates of
enzyme catalyzed
reactions.

Michaelis-Menten Kinetics
• 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 (V
max), substrate concentration [S] and
the Michaelis-Menten constant (km).

Michaelis-Menten Kinetics
• The dependence of initial reaction velocity on [S] and Km may be
illustrated by evaluating the Michaelis-Menten equation under three
conditions.
• (1) When [S] is much less than km, the term km + [S] is essentially
equal to km. Replacing Km + [S] with Km reduces equation to

When [S] is
much less
than km
• Since V max and km are both constants, their
ratio is a constant (k).
• In other words, when [S] is considerably below
km, V max is proportionate to k[S].
• The initial reaction velocity therefore is directly
proportionate to [S].

When [S] is much greater than km
• ,the term km + [S] is essentially equal to [S]. Replacing km + [S] with
[S] reduces equation to :
Thus, when [S] greatly exceeds km, the reaction velocity is
maximal (V max) and unaffected by further increases in
substrate concentration.

(3) When [S] = km
• Equation states that when [S] equals km, the initial velocity
is half-maximal.
• Equation also reveals that km can be determined
experimentally from—the substrate concentration at which
the initial velocity is half-maximal.

Linear Form of the Michaelis-Menten Equation
• The direct measurement of the numeric value of V max and therefore
the calculation of km often requires impractically high concentrations
of substrate to achieve saturating conditions.
• A linear form of the Michaelis-Menten equation circumvents this
difficulty and permits V max and km to be extrapolated from initial
velocity data obtained at less than saturating concentrations of
substrate.

Linear Form of the Michaelis-Menten Equation
invert
Factor
and simplify

Double reciprocal or Lineweaver-Burk plot
• Equation for a straight line, y = ax + b,
where y = 1/vi and x = 1/[S].
• A plot of 1/vi as y as a function of
1/[S] as x therefore gives a straight
line whose y intercept is 1/ V max and
whose slope is km / V max.
• Such a plot is called a double
reciprocal or Lineweaver-Burk plot

Carry home message
Enzyme is affected by:
❑Substrate concentration
❑Product concentration
❑Enzyme concentration
❑Temperature
❑pH
❑Time and
❑Presence of activators and inhibitors

Thank you

Factors affecting enzyme activity

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