This document discusses enzyme kinetics and inhibition. It begins by defining enzymes as protein catalysts and describing how they lower the activation energy of biochemical reactions. It then defines enzyme kinetics as the study of reaction rates catalyzed by enzymes. Key terms introduced include reaction rate, Michaelis-Menten equation, Vmax, and Km. The document describes different types of enzyme inhibitors - competitive, non-competitive, uncompetitive - and how they each affect the kinetic parameters Vmax and Km. Finally, it presents two sample problems analyzing kinetic data to determine the type of inhibition occurring.

1. Presented by/ Esraa M Mosalam
Assistant Lecturer of Biochemistry
Faculty of Pharmacy
Menoufia University
Enzyme
Kinetics

2. Introduction
 Enzymes are protein catalysts for biochemical
reactions in biological systems.
 All chemical reactions proceeds from initial substrate
to products consuming energy; this energy is called
energy of activation.
 Enzymes act by decreasing energy of activation
 They increase the rate of chemical reactions without
changing themselves.
2

3. Enzyme Kinetics
• Enzyme Kinetics
It is the study of the rate (velocity) of reactions catalyzed by
enzymes.
• Rate of reaction (turn over or velocity):
o Is the number of moles of substrate converted to product
per second (mol/s).
o The enzyme activity is measured by the rate of reaction.
• Michaelis-Menten Equation
Describe how reaction velocity varies with substrate
concentration.
3

4. Enzyme substrate interaction
4

5. Michaelis-Menten Equation
5

6. 6
Hyperbolic curve
Michaelis-Menten Equation

7. • vo (Initial velocity):
o This means that the rate of the reaction is measured
as soon as enzyme and substrate are mixed.
o The rate at which reaction proceeds is measured as
decrease in concentration of reactants (substrate) or
increase in concentration of products with time.
• Vmax:
Represents the maximum rate achieved by the system,
at maximum saturating substrate concentrations.
7
Michaelis-Menten Equation

8. Michaelis-Menten Equation
Km (Michaelis-Menten constant)
o Km is numerically equal to the substrate concentration
at which the reaction velocity is equal to ½Vmax.
o Km does not vary with the concentration of enzyme.
o Is characteristic of an enzyme and its particular
substrate, and reflects the affinity of the enzyme for
that substrate.
8

9. Indication of Km value
Large KmSmall Km
• Reflects a low affinity of
enzyme for substrate.
• Because a high concentration
of substrate is needed to half-
saturate the enzyme to reach
½Vmax.
• Reflects a high affinity of the
enzyme for substrate.
• Because a low concentration
of substrate is needed to half-
saturate the enzyme to reach
½Vmax.
9

10. Effect of substrate
concentration on reaction
velocities for two enzymes
10

11. Lineweaver-Burk Plot
11

12. Lineweaver-Burk plot
(double-reciprocal plot)
• When vo is plotted against [S], it is not always possible
to determine when Vmax has been achieved, because
of the gradual upward slope of the hyperbolic curve.
But …Why is the graph not linear?
12

13. Lineweaver-Burk plot
13

14. Significance of Lineweaver-Burk
plot
 Calculate Km
 Calculate Vmax
 Determine the mechanism of action of enzyme
inhibitors.
14

15. Enzyme Inhibition
15

16. Enzyme Inhibitors
• Inhibitors are substances that binds to an
enzyme and interfere with its activity. Thus,
reduce the rate of enzymatic reactions.
• It can prevent formation of enzyme substrate
(ES) complex or can prevent ES breakdown to
enzyme + product.
• E + S → ES Complex → Enzyme + Product.
• E + S + I → ESI or EI → decreased product or
no product formation.
16

17. Types of Enzyme Inhibitors
Enzyme
inhibitors
Reversible
Competitive
Non-competitive
Uncompetitive
Irreversible
Competitive
(active site
directed)
Allosteric
(suicide
inhibitors) 17

18. Types of Enzyme Inhibitors
Reversible
• Inhibitor binds to Enzyme
reversibly through non
covalent interactions.
• The activity of enzyme is
fully restored on removing
the inhibitor by dialysis.
• E + I ⇌ E I
Irreversible
• Inhibitor binds at or near
the active site of the
enzyme irreversibly, usually
by covalent bonds, so it
can’t dissociate from the
enzyme.
• Enzyme activity is not
regained on dialysis.
• E + I ⇌ E I
• Ex. Organophosphorus
insecticide irreversibly
inhibit acetylcholine
esterase.
18

19. Reversible
Inhibitors
19

20. Competitive Inhibition
• The inhibitor and substrate compete for the same
active site on the enzyme as a result of similarity in
structure.
• Inhibition is reversible by increasing substrate
concentration.
20

21. Competitive Inhibition
• Ex. the anti-gout drug
“Allopurinol” is similar in
structure to
“hypoxanthine”; substrate
for xanthine oxidase; an
intermediate step in the
catabolism of purines.
• Ex. the anti-cancer drug
“methotrexate” is similar in
structure to “dihydrofolate”;
substrate for dihydrofolate
reductase; an essential step
in biosynthetic pathway of
folic acid.
21

22. Kinetics of Competitive Inhibitor
Effect on Vmax:
 At a sufficiently high substrate concentration, the
reaction velocity reaches the Vmax observed in the
absence of inhibitor (Vmax not changed).
Effect on Km:
 A competitive inhibitor increases the apparent Km for a
given substrate.
 This means that, in the presence of a competitive
inhibitor, more substrate is needed to achieve ½Vmax.
22

23. 23

24. Noncompetitive Inhibition
• Noncompetitive inhibition occurs when the inhibitor
and substrate bind at different sites on the enzyme.
• These inhibitors can bind to enzyme or enzyme-
substrate complex.
• Not affected by increasing substrate concentration.
24

25. Noncompetitive Inhibition
• Ex. Cyanide combines with the iron in the
enzymes cytochrome C oxidase.
• Ex. Silver ions (heavy metal) react with -SH
groups in the side groups of cysteine residues
in the protein chain.
25

26. 26

27. Kinetics of Noncompetitive Inhibitior
Effect on Vmax:
 Noncompetitive inhibition cannot be overcome by
increasing the concentration of substrate. Thus,
noncompetitive inhibitors decrease the apparent Vmax of
the reaction.
Effect on Km:
 Noncompetitive inhibitors do not interfere with the
binding of substrate to enzyme. Thus, the enzyme shows
the same Km in the presence or absence of the
noncompetitive inhibitor.
27

28. 28

29. Uncompetitive Inhibition
• Inhibitor binds only to enzyme-substrate
complex.
• Binding site for inhibitor is created only upon
substrate binding.
• Not affected by increasing substrate
concentration.
29

30. Uncompetitive Inhibition
• Ex. Inhibition of placental alkaline
phosphatase (PALP) by phenylalanine.
• Ex. Sulfoxide & 3-butylthiolene-1-oxide are
uncompetitive inhibitors of liver
alcoholdehydrogenase (ADH).
30

31. Uncompetitive Inhibition
31

32. Kinetics of Uncompetitive
Inhibitor
Effect on Vmax:
 Uncompetitive inhibition decrease Vmax
because they interfere with substrate binding
and hamper catalysis in the ES complex.
Effect on Km:
 Uncompetitive inhibitors has no affinity for
free enzyme. Thus, they decrease the
apparent Km for a given substrate.
32

33. Kinetics of Uncompetitive
Inhibitor
33

34. Problems

35. Problem (1)
Salicylate (aspirin) inhibits the catalytic action of
glutamate dehydrogenase.
Plot the data two ways:
1) v vs [S]
2) 1/v vs 1/[S] on graph paper.
3) Estimate the Vmax and Km in the presence and absence of this
inhibitor.
4) From the data, can you determine the type of inhibition
(competitive or non-competitive)?
35

36. Problem (1)
36

37. 37

38. 1/V vs 1/S
1/v
(with inhibitor)
1/v
(No inhibitor)
1/[s]
12.54.80.67
1040.5
8.33.60.33
7.730.25
6.32.30.125
5.62.50.0625 38

39. 39

40. Problem (2)
Another type of inhibitor of glutamate
dehydrogenase was tested:
a) Do the same calculations on this inhibitor.
b) From the data, can you determine the type of
inhibition?
40

41. Problem (2)
Product per minute (nM)
6mM inhibitorNo inhibitorSubstrate (mM)
88 (0.88X102)139 (1.39X102)2
121 (1.21X102)179 (1.79X102)3
149 (1.49X102)213 (2.13X102)4
257 (2.57X102)313 (3.13X102)10
313 (3.13X102)370 (3.70X102)15
41

42. 42

43. 1/V vs 1/S
Velocity
1/V with inhibitor X10-31/V no inhibitor
X 10-3
1/S
11.4 (0.0114)(7) 0.0070.5
8.3 (0.0083)(5.6) 0.00560.33
6.7 (0.0067)(4.7) 0.00470.25
3.9 (0.0039)(3.2) 0.00320.1
3.2 (0.0032)(2.7) 0.0027o.067
43

44. 44

45. 45