Transcript
Page 1: Chapter 6.3:  Enzyme Kinetics

Chapter 6.3: Enzyme Kinetics

CHEM 7784

Biochemistry

Professor Bensley

Page 2: Chapter 6.3:  Enzyme Kinetics

CHAPTER 6.3Enzyme Kinetics

– description of enzyme kinetics by examining the Michaelis-Menten theory

Today’s Objectives: (To learn and understand the)

Page 3: Chapter 6.3:  Enzyme Kinetics

What is (are?) Enzyme Kinetics?

• Kinetics is the study of the rate at which compounds react

• Rate of enzymatic reaction is affected by– Enzyme– Substrate– Effectors– Temperature

Page 4: Chapter 6.3:  Enzyme Kinetics
Page 5: Chapter 6.3:  Enzyme Kinetics

How to Take Kinetic Measurements

Page 6: Chapter 6.3:  Enzyme Kinetics

Effect of Substrate Concentration

• Ideal Rate:

• Deviations due to:

–Limitation of measurements

–Substrate inhibition

–Substrate prep contains inhibitors

–Enzyme prep contains inhibitors

SK

SVv

m

][max

Page 7: Chapter 6.3:  Enzyme Kinetics

Plot V0 vs. [S]

• Michaelis-Menten Equation• Describes rectangular hyperbolic plot

Vo = Vmax [S] Km + [S]

Page 8: Chapter 6.3:  Enzyme Kinetics

Km = [S] @ ½ Vmax (units moles/L=M)(1/2 of enzyme bound to S)

Vmax = velocity where all of the enzyme is bound to substrate (enzyme is saturated with S)

Page 9: Chapter 6.3:  Enzyme Kinetics

1) Measurements made to measure initial velocity (vo). At vo very little product formed. Therefore, the rate at which E + P react to form ES is negligible and k-2 is 0. Therefore

Initial Velocity Assumption

E + S ES E + Pk1

k-1

k2

E S+ E S

k-2

E + P

Page 10: Chapter 6.3:  Enzyme Kinetics

Steady State Assumption

E + S ES E + Pk1

k-1

k2

Steady state Assumption = [ES] is constant. The rate of ES formation equals the rate of ES breakdown

E S+ E S E + P

Page 11: Chapter 6.3:  Enzyme Kinetics

E + S ESk1

E S+ E S

Rate of ES formation

Rate = k1 [E] [S]

Page 12: Chapter 6.3:  Enzyme Kinetics

ES E + Pk2

E S E + P

ES E + Sk-1

E S+E S

Rate of ES breakdown

Rate = (k2 [ES]) + (k-1[ES])

Rate = [ES](k2 + k-1)

Page 13: Chapter 6.3:  Enzyme Kinetics

Therefore………if the rate of ES formation equals the rate of ES breakdown

1) k1[E][S] = [ES](k-1+ k2)

2) (k-1+ k2) / k1 = [E][S] / [ES]

3) (k-1+ k2) / k1 = Km (Michaelis constant)

Page 14: Chapter 6.3:  Enzyme Kinetics

What does Km mean?

1. Km = [S] at ½ Vmax

2. Km is a combination of rate constants describing the formation and breakdown of the ES complex

3. Km is usually a little higher than the physiological [S]

Page 15: Chapter 6.3:  Enzyme Kinetics

What does Km mean?

4. Km represents the amount of substrate required to bind ½ of the available enzyme (binding constant of the enzyme for substrate)

5. Km can be used to evaluate the specificity of an enzyme for a substrate (if obeys M-M)

6. Small Km means tight binding; high Km means weak binding

Glucose Km = 8 X 10-6

Allose Km = 8 X 10-3

Mannose Km = 5 X 10-6

HexokinaseGlucose + ATP <-> Glucose-6-P + ADP

Page 16: Chapter 6.3:  Enzyme Kinetics

What does kcat mean?

E + S ES E + Pk1

k-1

kcat

Page 17: Chapter 6.3:  Enzyme Kinetics
Page 18: Chapter 6.3:  Enzyme Kinetics

What does kcat/Km mean?

Page 19: Chapter 6.3:  Enzyme Kinetics
Page 20: Chapter 6.3:  Enzyme Kinetics

Aren’t Enzymes Kinetics Fun?!

• The final form of M-M equation in the case of a single substrate is

• kcat (turnover number): how many substrate molecules can one enzyme molecule convert per second

• Km (Michaelis constant): an approximate measure of substrate’s affinity for enzyme

][

]][[

SK

SEkv

m

totcat

Page 21: Chapter 6.3:  Enzyme Kinetics

Limitations of M-M

1. Some enzyme catalyzed rxns show more complex behaviorE + S<->ES<->EZ<->EP<-> E + P

With M-M can look only at rate limiting step

2. Often more than one substrate E+S1<->ES1+S2<->ES1S2<->EP1P2<-> EP2+P1<-> E+P2

Must optimize one substrate then calculate kinetic parameters for the other

3. Assumes k-2 = 0

4. Assume steady state conditions

Page 22: Chapter 6.3:  Enzyme Kinetics
Page 23: Chapter 6.3:  Enzyme Kinetics
Page 24: Chapter 6.3:  Enzyme Kinetics

BB

B

BB B

B

0

0.05

0.1

0.15

0.2

0.25

0 1 2 3 4 5 6 7 8 9 10

Vo

[S]

[S] Vo0.5 0.0750.75 0.092 0.1524 0.1966 0.218 0.21410 0.23

V max

KmKm ~ 1.3 mM

Vmax ~ 0.25

Page 25: Chapter 6.3:  Enzyme Kinetics

[S] Vo2.000 13.3331.333 11.1110.500 6.5790.250 5.1020.167 4.7620.125 4.6730.100 4.348

B

B

B

BBBB

B0

2

4

6

8

10

12

14

-1 -0.5 0 0.5 1 1.5 2

Vo

[S]

-1/Km = -0.8Km = 1.23 mM1/Vmax = 4.0Vmax = 0.25

1/ 1/


Top Related