Enzyme kinetics is the study of how fast enzyme-catalyzed reactions occur. The Michaelis-Menten equation describes the relationship between substrate concentration and reaction rate. It defines terms like Vmax, the maximum reaction rate when the enzyme is saturated with substrate, and Km, the substrate concentration needed to reach half Vmax. Factors like temperature, pH, and enzyme/substrate concentrations can impact kinetic parameters and reaction rates.

1. ENZYME KINETICS

2. • Chemical kinetics is the branch of chemistry
which addresses "how fast do reactions go?"
• Enzymes are the catalysts of biological systems
and are extremely efficient and specific as
catalysts.
• An enzyme accelerates the rate of a reaction
• Enzymes are highly specific.
CHEMICAL KINETICS

3. • A chemical mechanism is the chemical pathway
of conversion of S → P, including the structures
of any intermediates

4. • What we want to be able to determine
– Maximum velocity
– Substrate affinity
– Inhibitor affinity
• What it can tell us
– Flow through metabolic pathways
– Utilization of substrates
• What can we do with the information
– Control and manipulate metabolic events

5. STUDY OF ENZYME KINETICS IS USEFUL FOR MEASURING
– Concentration of an enzyme in a mixture
– Its purity
– Its specificity for different substrates
– Comparison of different forms of the same enzyme
– Effects of inhibitors
- Mechanism, structure of active site

6. ENZYME KINETICS
• The study of rate of the reaction and how it
changes in response to change in experimental
parameters
• These studies include measuring rates of the
enzyme-catalyzed reactions
[S]
Vmax/2
Km
v = Vmax = kcat [E]o

7. • In 1913 Michaelis-Menten derive an equation
which gives the relationship between the [S] &
velocity of the reaction.
• E is the enzyme, S is the "substrate" ES is an
enzyme-substrate complex.
Leonor Michaelis
(1875-1940)
Maud L. Menten
(1879-1960)
MICHAELIS-MENTEN

8. • E+S ES
K1
K-1
ES E+P
K2
K-2
Equation 1
Vo = K2 [ES] Equation 2
• ES is not easily measured thus the term [Et] is
introduced which is the sum of Free enzyme +
substrate bound enzyme
[Et] = [E] + [ES]
[E] = [Et] – [ES]

9. Rate of ES formation = K1([Et]-[ES]) [S] Equation 3
Rate of ES breakdown = K-1([ES]+K2[ES]) Equation 4
K1 ([Et]) – [ES])[S] = K-1 [ES] + K2 [ES] Equation 5
K1 [Et][S] - K1 [ES] [S] = (K-1 + K2) [ES] Equation 6
Step 1 : The rate of formation and break down of ES are
determined by the steps governed by the rate constant
Step 2 : An assumption is made that the initial rate of the
reaction reflects a steady state

10. K1 [Et] [S] = (K1 [S] + K-1 + K2) [ES] Equation 7
[ES] = K1 [Et] [S] Equation 8
K1 [S] + K-1 + K2
[ES] = [Et] [S]
Equation 9
[S] + (K2 + K-1) / K1
Adding the term K1 [ES][S] to both sides of the equation
Solving for ES
Dividing by K1

11. The term (K2 + K-1) / K1 is defined as Km
i.e., [ES] = [Et] [S]
Km + [S]
Equation 10
Vo can be express in terms of [ES]
Vo = K2 [ES]
Vo = K2 [Et] [S] Equation 11
Km + [S]
Vo = Vmax [S]
Km + [S]
Maximum velocity occur when the enzyme is saturated
[ES] = [Et]. Vmax = K2[Et]

12. RECIPROCAL OF MICHEAL MENTEN’S EQUATION
Vo is exactly half of the Vmax
Vmax/2 = Vmax [S] / Km + [S]
On dividing by Vmax
½ = [S] / Km + [S]
Solving for Km, we get Km + [S] = 2 [S]
Km = [S]

13. LINEWEAVER BURK RECIPROCAL PLOT
• It is difficult to determine the limitting value of V
(i.e., Vmax) directly from a plot of V against [S]
• Reciprocal of the MM equation give a straight line
plot
1/V0 = Km + [S]
Vmax [S]
1/Vo = Km + [S]
Vmax [S] Vmax [S]
1/Vo = Km + 1
Vmax [s] Vmax

14. •1/Vo is ploted against 1/[S] a straight line is obtained.
• Slope - Km/Vmax
•An intercept 1/ Vmax on the 1/ Vo axis
•-1/Km on the 1/[S] axis

15. HALDANE AND BRIGGS EQUATION
• In 1925 Briggs and Haldane, came up with a
more generally applicable assumption.
• They assumed that the concentration of ES
does not change with time. This is the Steady
State Assumption (Briggs and Haldane).
[E] + [S] [ES] [P] + [E]
k2
k-1
k1

16. EFFECT OF ENZYME CONCENTRATION
• As the enzyme concentration increases the rate
of enzyme activity increases up to a level where
it becomes constant.
• More the enzymes are available, the more
substrates are broken in less time.

17. •Then becomes constant as the substrate acts as a
limiting factor, which means that there are not
enough substrates to be broken down compared to
the number of enzymes.

18. EFFECT OF PH
• If the pH is too acidic or too basic for an enzyme,
its hydrogen bonds begin to break, causing its
active site to change its shape.
• An altered active site can’t bind with its
substrate so enzyme activity decreases.

19. • If the pH is too unfavorable then covalent bonds
can break, causing the enzyme to denature.

20. EFFECT OF TEMPERATURE
• The temperature increases there is more
movement of molecules and therefore more
collisions between enzymes and substrates – so
the enzyme activity increases.
• There is a limit to which enzyme activity can
increase because at a certain temperature an
enzyme will denature

21. EFFECT OF SUBSTRATE CONCENTARTION
• [S] changes due to the conversion of substrate
to product.
• Measure the initial rate of the reaction
designated V0 ( Initial velocity), when [S] is
much greater than the concentration of enzyme

22. SIGNIFICANCE OF Km AND Vmax
• The Km values of enzymes range widely. Km
values lies between 10-1 & 10-7 M
• The Km value of an enzyme depends on the
particular substrate & also environmental
conditions such as pH, temperature & ionic
strength
• Km is the concentration of substrate at which
half the active sites are occupied
• At lower Km affinity of enzyme is more and at
higher Km affinity of enzyme is low

23. • Km is related to the rate constants of
individual steps in the catalytic scheme
• If K-1 is greater than K2 i.e., dissociation of ES
complex is more rapid than the formation of E
& P then
Km = K-1 / K1
• The dissociation constant of ES complex is
given by
KES = [E] [S] = K-1
[ES] K1
E+S K1 ES K2 E+P
K-1

24. • In some cases the ES complex doesn't breakdown
directly to form free enzyme and product but
they for EP complex. Hence these is an rate
limiting step
Vmax = K3 [Et]
• Turn over number (Kcat) is defined as the number
of substrate converted to product by single
enzyme in a unit of time when enzyme is fully
saturated with substrate.
Vmax = Kcat [Et]
Vo = Vmax [S] / Km + [S]
Vo = Kcat [Et] / Km = [S]
E+S K1 ES K2 EP E+P
K-1 K-2