1. Allosteric Enzymes
• Allosteric enzymes have one or more allosteric sites
• Allosteric sites are binding sites distinct from an enzyme’s active site or substrate-binding site
• Molecules that bind to allosteric sites are called effectors or modulators
• Binding to allosteric sites alters the activity of the enzyme. This is called cooperative binding. Allosteric enzymes display sigmoidal plot of Vo vs [S]
• Effectors may be positive or negative
• Effectors may be homotropic or heterotropic
• Regulatory enzymes of metabolic pathways are allosteric enzymes (eg: feedback inhibition)

2. Allosteric enzymes Allosteric enzymes tend to be
multi-sub unit proteins
The reversible binding of an
allosteric modulator (here a
positive modulator M) affects
the substrate binding site

4. Mechanism and Example of Allosteric Effect
Kinetics
Cooperation
Models
Allosteric site
R = Relax
(active)
[S] vo
Homotropic
(+)
Concerted
(+)
Allosteric site
[S] vo
(+) A
Heterotropic
(+)
Sequential
(+) X
T = Tense
(inactive)
[S] vo I
Heterotropic
(-)
Concerted
(-)
(-) X X

5. Enzyme Inhibitors
• Specific enzyme inhibitors regulate enzyme activity and help us understand mechanism of enzyme action. (Denaturing agents are not inhibitors)
• Irreversible inhibitors form covalent or very tight permanent bonds with aa at the active site of the enzyme and render it inactive. 3 classes: groupspecific reagents, substrate analogs, suicide inhibitors
• Reversible inhibitors form an EI complex that can be dissociated back to enzyme and free inhibitor. 3 groups based on their mechanism of action: competitive, non-competitive and uncompetitive.

6. Enzyme Inhibition

7. Competitive inhibitors
• Compete with substrate for binding to enzyme
• E + S = ES or E + I = EI . Both S and I cannot bind enzyme at the same time
• In presence of I, the equilibrium of E + S = ES is shifted to the left causing dissociation of ES.
• This can be reversed / corrected by increasing [S]
• Vmax is not changed, KM is increased by (1 + I/Ki)
• Eg: AZT, antibacterial sulfonamides, the anticancer agent methotrexate etc

8. Competitive Inhibition

9. Kinetics of competitive inhibitor
Increase [S] to
overcome
inhibition
Vmax attainable,
Km is increased
Ki =
dissociation
constant for
inhibitor

10. V max unaltered, Km increased

11. Non-competitive Inhibitors
• Inhibitor binding site is distinct from substrate binding site. Can bind to free enzyme E and to ES
• E + I = EI, ES + I = ESI or EI + S = ESI
• Both EI and ESI are enzymatically inactive
• The effective functional [E] (and [S]) is reduced
• Reaction of unaffected ES proceeds normally
• Inhibition cannot be reversed by increasing [S]
• KM is not changed, Vmax is decreased by (1 + I/Ki)

12. Mixed (Noncompetitive) Inhibition

13. Kinetics of non-competitive inhibitor
Increasing [S] cannot
overcome inhibition
Less E available,
V max is lower,
Km remains the same
for available E

14. Km unaltered, V max decreased

15. Uncompetitive Inhibitors
• The inhibitor cannot bind to the enzyme directly, but can only bind to the enzyme-substrate complex.
• ES + I = ESI
• Both Vmax and KM are decreased by (1+I/Ki).

16. Uncompetitive Inhibition

17. Substrate Inhibition Caused by high substrate concentrations
E + S ES E + P
Km’ k2
KS1 + S
ES2

18. Substrate Inhibition
At low substrate concentrations [S]2/Ks1<<1 and inhibition is not observed
Plot of 1/v vs. 1/[S] gives a line
Slope = K’m/Vm
Intercept = 1/Vm

19. Substrate Inhibition
At high substrate concentrations, K’m/[S]<<1, and inhibition is dominant
Plot of 1/v vs. [S] gives a straight line
Slope = 1/KS1 · Vm
Intercept = 1/Vm

20. 1/V
I>0
I=0
1/Vm
-1/Km
-1/Km,app
1/[S]
1/V
I>0
I=0
1/Vm
-1/Km
-1/Km,app
1/[S]
1/Vm,app
Competitive
Uncompetitive
1/V
I>0
I=0
1/Vm
-1/Km
1/[S]
1/Vm,app
1/V
1/Vm
-1/Km
1/[S]
Non-Competitive
Substrate Inhibition

21. Enzyme Inhibition (Mechanism)
Competitive
Non-competitive
Uncompetitive E
Substrate E X
Cartoon Guide
Compete for
active site
Inhibitor
Different site
E + S → ES → E + P + I ↓ EI ← ↑
E + S → ES → E + P
I I
↓ ↓
EI + S →EIS ← ↑
E + S → ES → E + P + I ↓
EIS ← ↑
Equation and Description
[I] binds to free [E] only,
and competes with [S];
increasing [S] overcomes
Inhibition by [I].
[I] binds to free [E] or [ES]
complex; Increasing [S] can
not overcome [I] inhibition.
[I] binds to [ES] complex
only, increasing [S] favors
the inhibition by [I].

22. Enzyme Inhibition (Plots)
Competitive
Non-competitive
Uncompetitive
Vmax
Vmax Km
[S], mM
Vmax
[S], mM vo vo
Direct Plots
Double Reciprocal
Vmax’ I
Km’
Vmax’ I I Km Km
Km’
[S], mM
= Km’
Vmax unchanged
Km increased
Vmax decreased
Km unchanged
Both Vmax & Km decreased
1/[S]
1/Km
1/vo
1/ Vmax I
1/vo
1/ Vmax
1/[S]
1/Km
1/[S]
1/Km
1/ Vmax
1/vo I
Intersect
at X axis I
Two parallel
lines
Intersect
at Y axis

23. Factors Affecting Enzyme Kinetics

24. Effects of pH - enzymes have ionic groups on their active sites.
- on enzymes
- enzymes have ionic groups on their active sites.
- Variation of pH changes the ionic form of the active sites.
- pH changes the three-Dimensional structure of enzymes.
- on substrate
- some substrates contain ionic groups
- pH affects the ionic form of substrate
affects the affinity of the substrate to the enzyme.

25. Effects of Temperature
Reaction rate increases with temperature up to a limit
Above a certain temperature, activity decreases with temperature due to denaturation
Denaturation is much faster than activation
Rate varies according to the Arrhenius equation
Where Ea is the activation energy (kcal/mol)
[E] is active enzyme concentration

26. Factors Affecting Enzyme Kinetics
Temperature
- on the rate of enzyme catalyzed reaction
k2=A*exp(-Ea/R*T)
T k2
- enzyme denaturation T
Denaturation rate:
kd=Ad*exp(-Ea/R*T)
kd: enzyme denaturation rate constant;
Ea: deactivation energy

27. REFERENCES
Michael L. Shuler and Fikret Kargı, Bioprocess Engineering: Basic Concepts (2 nd Edition),Prentice Hall, New York, 2002.
1. James E. Bailey and David F. Ollis, Biochemical Engineering Fundementals (2 nd Edition), McGraw-Hill, New York, 1986.

28. www-nmr.cabm.rutgers.edu/academics/biochem694/2005BioChem412/Biochem.412_2005_Lect.18.ppt –
juang.bst.ntu.edu.tw/BCbasics/Animation.htm - 37k – –