Problem Set #4:  Enzyme Kinetics

1) The enzyme lactate dehydrogenase catalyzes the reaction:

NADH absorbs light at 340 nm , but NAD+ does not. Suggest an experimental method for following the rate of this reaction, assuming that you have a spectrophotometer capable of measuring absorbances at this wavelength.

2) Show graphically the dependence of reaction velocity on substrate concentration for an enzyme that follows Michaelis-Menton kinetics and an allosteric enzyme.

3) For and enzyme that displays Michaelis-Menton kinetics, what is the reaction velocity, V (as a percentage of Vmax), observed at:

4) How is the turnover number of an enzyme related to Vmax ?

5) Fumarase catalyzes the hydration of fumarate to L-malate via the following reaction:

The enzyme is composed of four identical subunits and has a molecular weight of 194,000 Da. The data in the table below were obtained when fumarate was used as a substrate and the initial rates of hydration were measured at pH 5.7 and 25°C with an enzyme concentration of 2 x. 10-6M. Plot the data in Lineweaver-Burk form and determine Vmax, kcat, and Km for fumarase under these conditions.
 
[Fumarate] 

(mM)
Rate of Product Formation 

(mmole L-1 min.-1)
2.0
2.5
3.3
3.1
5.0
3.6
10.0
 4.2

6) A hypothetical enzyme has an active site with an effective pKa' value of 5. Draw pH-rate profiles for the following possibilities:

7) Draw a Lineweaver-Burk plots for an enzyme for which the following data are available.
 
[S] 

(mM)
V, no inhibitor 

(mmol min-1) 
V, with inhibitor 

(mmole min-1) 
3.0
4.58
3.66
5.0
6.40
5.12
7.0
7.72
6.18
9.0
8.72
6.98
11.0
9.50
7.60

What are the Km and Vmax values for the inhibited and uninhibited enzymes? Is the inhibitor competitive or non-competitive?

8) Ribonuclease catalyzes the hydrolysis of ribonucleic acid (RNA). The active site of the enzyme contains two histidine residues, whose participation is shown below:


 

The pH-rate profile is bell-shaped, with inflection points at approximately pH 5.8 and pH 6.2. How is the pH-rate profile related to the mechanism for ribonuclease?


Solutions to Problem Set 4:
 
Problem #1

Problem #2

Problem #3

Problem #4

Problem #5

Problem #6

Problem #7

Problem #8