Enzyme Kinetics

1
Lecture 11

• Enzyme Kinetics

2
Assays
3
Assays
4
Steady State Kinetics
Steady state ES constant
Pre-steady state ES builds
5
Typical Enzyme

• Reaction velocity vs. S
• Saturation kinetics

6
Typical Enzyme

• Can be explained by ES complex
• 1913 Michaelis and Menten

7
Does Equation Fit the Facts?
8
Relationship of k2 and k-1

• Michaelis-Menten mechanism (k2 ltlt k-1)
• Haldane case (k2 gt k-1)

9
Haldane Case
10
Michaelis-Menten vs. Haldane
11
Determing Km From a Graph
12
Vmax

• Reflects rate-limiting step at saturation
• For M-M Mechanism Vmax k2 Et
• For
• Vmax k3 Et

13
General Terms

• kcat Rate constant for rate-limiting step
• k2 or k3, etc
• Turnover number
• Vmax kcat Et
• Vo

14
Comparing Enzymes

• To compare enzymes, both kcat and Km are needed
• When S ltlt Km
• Vo
• limited to 108 M-1 s-1 by diffusion
• Specificity constant

15
Obtaining Km and Vmax

• Double-reciprocal plot
• Lineweaver-Burk plot

16
Obtaining Km and Vmax

• Eadie-Hofstee plot

17
Mechanistic Information

• Ordered BiBi
• Random bi-bi

18
Lineweaver-Burk Plot of Bisubstrate Reactions
Enzyme reaction involving a ternary complex
Have a point where lines intersect to the left
of the 1/vo axis
At this point
19
Mechanistic information

• Ping-Pong reactions (no ternary complex)

20
Bisubstrate Reactions
Enzyme reaction in which no ternary complex is
formed.
Slope
Intercept
21
Competitive inhibition

• A competitive inhibitor affects Km, but not Vmax

22
Competitive Inhibition

• Lineweaver-Burk plot

Slope changes Y intercept does not
Vmax does not change
23
Competitive Inhibition

• Eadie-Hofstee plot

24
Uncompetitive Inhibition

• I lowers Vmax and Km
• is unchanged

25
Noncompetitive Inhibition
26
Mixed Inhibition