Problem Set 3

 

 

 

1)      You are studying an enzyme, Glucose Isomerase, which converts glucose to fructose. To study the kinetics of the enzyme, you mix a small amount of enzyme with various concentrations of glucose and measure the reaction rate. You obtain the following results. [S] = [glucose], v=velocity.

 

[S] mM

v mM-min-1

5

22

10

39

20

65

50

102

100

120

200

135

 

a.       Estimate Vmax and Km for the data by plotting v as a function of [S]

 

 

b.      Estimate Vmax and Km for the data by plotting a Lineweaver-Burke plot

 

 

c.       Estimate Vmax and Km for the data by plotting an Eadie-Hofstee plot

 

 

2)      In your experiment, the total concentration of Glucose Isomerase is 1 nM.

a.       How many reactions can an enzyme catalyze in 1 minute? (i.e. what is kcat)

 

 

b.      Calculate kcat/Km for the enzyme (for kcat/Km, kcat is measured in sec-1, not min-1)

 

c.       Based on our discussion of enzyme efficiency and catalytic perfection, is this a very efficient enzyme?

 

 

 

3)      You also study the kinetics of butyrylcholinesterase, an engineered enzyme for the U.S. Army which deactivates organophosphorous nerve gases such as sarin. The Whamistani army has developed a compound which inhibits butyrylcholinesterase. You incubate the enzyme with various concentrations of the substrate, sarin, and measure the reaction velocity with (V+) and without (V-) the inhibitor, getting the following results:

 

S (mM)

V- (mM/min)

V+ (mM/min)

1.25

1.72

0.98

1.67

2.04

1.17

2.5

2.63

1.47

5

3.33

1.96

10

4.17

2.38

 

 

a.                   What kind of inhibitor is the Whamistani compound? (Plot the data and tell me the answer)

 

 

 

b.                  What are kcat and Km Vmax with and without inhibitor

 

4)      The same enzyme as in Problem 3 is analyzed in the presence of another inhibitor at two inhibitor concentrations (3 mM and 5 mM) with the following results.  

 

 

S

V - I

V + 3mM I

V + 5mM I

1.25

1.72

1.25

1.01

1.67

2.04

1.54

1.26

2.5

2.63

2

1.72

5

3.33

2.86

2.56

10

4.17

3.7

3.49

 

 

a)      What type of inhibitor is the second compound?

 

5)      The following data are obtained for the steady state kinetics of yet another enzyme-catalyzed reaction:

 

[S] mM

v mM/sec

0.250

0.260

0.330

0.450

0.500

0.920

0.750

1.800

1.000

2.500

2.000

4.100

4.000

4.800

 

a)                  Does this enzyme exhibit Michaelis-Menten kinetics?

 

 

b)                  What is Vmax?

 

 

6)      Enzymes lower the activation energy, DG for a reaction. What effect does this have on the equilibrium constant, Keq?