FUNDAMENTALS OF BIOCHEMISTRY ASSIGNMENT-
The following table lists data obtained from three kinetic experiments on an enzyme in the absence, and presence of two different inhibitors, A and B. The amount of enzyme present in each experiment was 5 µg in 10mL of reaction.
|
Substrate Concentration (mM)
|
Initial velocity (mM min-1) no inhibitor
|
Initial velocity (mM min-1) inhibitor A
|
Initial velocity (mM min-1) inhibitor B
|
|
1.25
|
1.72
|
0.98
|
1.01
|
|
1.67
|
2.04
|
1.17
|
1.26
|
|
2.50
|
2.63
|
1.47
|
1.72
|
|
5.00
|
3.33
|
1.96
|
2.56
|
|
10.00
|
4.17
|
2.38
|
3.49
|
(a) Use a suitable method to graph these data and determine Vmax and Km in the absence of any inhibitor, and the apparent Vmax and apparent Km in the presence of inhibitor A and inhibitor B.
(b) What types of inhibitors are A and B? Explain your answer.
(c) Can addition of extra substrate overcome the inhibition by either A or B? Why or why not?
(d) Given that the molecular weight of the enzyme is 35000 daltons (g/mol), determine the turnover number of the enzyme in the absence of inhibitors?
(e) Has this enzyme attained "catalytic perfection"? Explain your answer?
(f) The active site of this enzyme has an Aspartate residue that acts as a nucleophile and a Histidine residue that acts as a general acid catalyst. Draw the expected pH rate profile for the enzyme. Explain your answer.