ENZYMOLOGY
Prof. Dr. Rasha El
Sayed
Al-Azhar University
Enzyme Kinetics
• Introduction to Enzyme Kinetics
• Rate of a reaction
• Order of a reaction
• Michaelis-Menten equation
ENZYMES KINETICS
• Enzyme kinetics is the study of the rate of chemical
reactions (velocity) that are catalyzed by enzymes.
• Velocity (v) is assessed by the rate of change of
substrate to product per unit time.
• In enzyme kinetics, the reaction rate is measured and
the effects of variation in the conditions of the reaction
are investigated.
ENZYMES KINETICS
Importance
1-Drug Discovery and Development: Kinetic data help
determine how effectively a drug inhibits a target enzyme.
2-Industrial
Biocatalysis:
It
optimizes
bioreaction
performance, enabling more efficient production processes.
3-Metabolic Engineering: It is used to understand reaction
fluxes and how organisms adapt to environmental changes.
4-Understanding Disease: It aids in studying enzyme
dysfunction related to diseases
ENZYMES KINETICS
Transition state
(Ea)
Free energy
Vary fom reaction
to other
The free energy of a chemical
reaction is the amount of energy
released in the conversion of
reactants to products under
standard conditions.
ENZYMES KINETICS
• All chemical reactions have an activation energy (Ea).
• The activation energy is the minimum amount of energy needed for a
reaction to occur.
• Enzymes lower the Ea of a particular reaction (do this because they have a
high affinity for a transition state). E + S  ES
• Enzymes assist in the reaction, so less energy is required. This means the
reaction can occur more easily. This speeds up the rate of the reaction as
it allows the product to be formed faster.
ENZYMES KINETICS
• An enzyme has a high affinity for the transition state (even higher than for its
substrate). Therefore, when the substrate binds, it is quickly forced into the
transition state (It is a state that exists between the substrate and the
product). The enzyme is said to facilitate the formation of the transition
state.
• The transition state has a high energy, making it very unstable. It can only exist
transiently. The transition state spontaneously turns into the more stable
product with lower energy. The enzyme has a low affinity for the product and
so the product is released.
• Then, the rate of the enzymatic reaction is directly proportional to the
concentration of ES.
ENZYMES KINETICS
The rate of the enzymatic reaction is directly proportional to the
concentration of ES.
• The concentration of ES changes as the reaction progresses.
• Therefore, the rate of product formation also changes over time.
• When the reaction reaches equilibrium (steady state) the concentration of
ES (and therefore the rate) remains relatively constant.
ENZYMES KINETICS
• Reaction Kinetics
• When an enzyme is added to a lot of substrate, the reaction occurs in
three stages with distinct kinetics: of ES
The pre-steady state phase is very short as equilibrium is reached within microseconds. If you measure the rate in the first
few seconds of a reaction (V0), you are actually measuring the steady state. This is the rate used in Michaelis-Menten Kinetics.
Effect of [S] on enzymatic reactions
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; Calmost unchanged).
Maximum activity
All enzymes
combined
with S
enzyme activity
increases
FIG. Effect of substrate concentration
on enzyme velocity (A- linear; B-curve;
C-almost unchanged).
Effect of [S] on enzymatic reactions
FIG. Effect of substrate
concentration on enzyme
velocity (A- linear; B-curve;
C-almost unchanged).
Effect of [S] on enzymatic reactions
Vmax = the maximum rate (velocity) of reaction when all enzyme active
sites are saturated with substrate (all of the enzyme is bound to
substrate; i.e., (enzyme is saturated with S); unit for Vmax is
moles/time
Km = the substrate concentration that
gives half maximal velocity. [S] when
v is equal to ½ Vmax , Unit for Km is
concentration (moles/L = M)
FIG. Effect of substrate concentration on enzyme velocity (A- linear; Bcurve; C-almost unchanged).
Mixed/Order
reaction: As
increases,
the reaction
order
transitions
from firstorder to zeroorder
Order of enzymatic reactions
Zero-order reaction: When the [S] is much
greater than Km, the rate of reaction is
independent of substrate concentration.
First-order reaction: the velocity of the reaction
is proportional to the substrate concentration
(i.e. [S] is less than Km)
FIG. Effect of substrate concentration on enzyme velocity (A- linear; Bcurve; C-almost unchanged).
Michaelis-Menten Kinetics Model
Role of enzyme catalysis (V0): number of moles of product / second
• Michaelis-Menten explained kinetic characteristics
E = Enzyme
S = Substrate
P = Product
ES = Enzyme-Substrate complex
k1 = rate constant for the association of E with S to form ES
k2 = rate constant for the dissociation of ES to E and S
k3 = rate constant for the conversion of ES to E and the product
Michaelis constant is given by the formula:
Michaelis-Menten Kinetics Model
The equation concerns the steady state of an enzymatic reaction
with one substrate, is given by:
where v = Measured velocity,
Vmax = Maximum velocity,
S = Substrate concentration,
Km = Michaelis – Menten
constant.
Let us assume that the measured velocity (v) is equal to ½ Vmax.
Then the equation may be substituted as follows:
Michaelis-Menten Kinetics Model
The equation concerns the steady state of an enzymatic reaction
with one substrate, and is given by:
Where,
Vmax = Maximum velocity,
S = Substrate concentration,
Km = Michaelis – Menten
constant.
Michaelis-Menten Kinetics Model
Km –Km is a measure of the affinity an enzyme has for its
substrate, as a lower Km means that less of the substrate is required
to reach half of Vmax.
Km is inversely proportional to the affinity of the substrate to the enzyme
(i.e., the higher the Km, the lower the affinity; The lower the Km, the higher the affinity
Michaelis-Menten Kinetics Model
Km –
1- It is a constant and characteristic feature of a given enzyme
(comparable to a thumb impression or signature).
2- It is a representative for measuring the strength of ES complex.
a. Small Km: A numerically small (low) Km reflects a high affinity of the
enzyme for substrate, because a low concentration of substrate is needed to
half-saturate the enzyme, that is, to reach a velocity that is 1⁄2Vmax.
b. Large Km: A numerically large (high) Km reflects a low affinity of enzyme
for substrate because a high concentration of substrate is needed to halfsaturate the enzyme.
Km is inversely proportional to the affinity of the substrate to the enzyme
(i.e., higher the Km, lower the affinity; lower the Km, higher the affinity