1. Explain what is meant by the order of the reaction, using the reaction below as an example.

What is the reaction order for each reactant? For the overall reaction? (Consider the forward and the reverse reaction.) A+B 2C

8. Assume that an enzyme-catalyzed reaction follows Michaelis-Menten kinetics with KM of 1M. The initial velocity is 0.1M/min at 10mM substrate. Calculate the initial velocity at 1mM, 10M and 1M substrate. If the substrate concentration increased to 20mM, would the initial velocity double? Why or why not? 9. If the KM for an enzyme is 1.0 x 10-5 M and KI of a competitive inhibitor enzyme of the enzyme is 1.0 x 10-6 M, what concentration of inhibitor would be necessary to lower the reaction rate of 10 when the substrate concentration is 1.0 x 10-3 M? 1.0 x 10-5M? 1.0 x 10-6 M? 10. Assume that an enzyme-catalyzed reaction follows the scheme shown: k k
1 3

2. In a first-order reaction a substrate is converted to product so that 87% of the substrate is converted in 7 min. Calculate the first-order rate constant. In what time is 50% of the substrate converted to product? 3. Prove that the KM equals the substrate concentration at one-half maximal velocity? 4. The Michaelis constant KM is frequently equated with KS, the [ES] dissociation constant.

However, there is usually a disparity between those values. Why? Under what conditions are KM and KS equivalent? 5. When quantifying the activity of an enzyme, does it matter if you measure the appearance of a product or the disappearance of a reactant? 6. An enzyme was assayed with the substrate concentration of twice KM value. The progress curve of the enzyme (product produced per minute) is shown. Give two possible reasons why the progress curve becomes nonlinear. Product formed

E+S

k2

ES

k4

E+P

where k1 = 109M-1s-1, k2 = 105s-1, k3 = 102s-1, k4 = 107M-1s-1, and [Et] is 0.1nM. Determine the value of each of the following. a. Km b. Vmax c. Turnover number d. Initial velocity when [S]o is 20M. 11. A colleague has measured the enzymatic activity as a function of reaction temperature and obtained the data shown in this graph. He insists on labeling point A as the temperature optimum for the enzyme. Try, tactfully, to point out the fallacy on that interpretation.

Time 7. What is the steady-state approximation and under what conditions is it valid?

Enzy mati c activ ity

30

50

Temperature, oC

70

12. You have isolated a titrimetric NAD+ dependent dehydrogenase. You incubate this enzyme with iodoacetamide in the absence or presence of NADH (at 10 times the KM concentration), and you periodically remove aliquots of the enzyme for the activity measurements and amino acid composition analysis. The results of the analyses are shown in the table.
(No NADH Present) Time (min) 0 15 30 45 60 Activity (U/mg) 1000 560 320 180 100 20 18.2 17.3 16.7 16.4 12 11.4 10.8 10.4 10.0 His Cys Time (min) 0 15 30 45 60 (NADH Present) Activity (U/mg) 1000 975 950 925 900 20 20 20 19.8 19.6 12 11.4 10.8 10.4 10.0 His Cys

to a final concentration of 0.01nM. Derive KM, Vmax, kcat, and the specificity constant. 14. You measured the initial velocity of an enzyme in the absence of inhibitor and with inhibitor A or inhibitor B. In each case, the inhibitor is present at 10M. The data are shown in the table.
Velocity [S] (mM) (Ms-1) x 107 Velocity (Ms-1) x 107 Velocity (Ms-1) x 107 Inhibited B

Uninhibited

Inhibited A

0.333 0.4 0.5 0.666 1.0 2.0

1.65 1.86 2.13 2.49 2.99 3.72

1.05 1.21 1.43 1.74 2.22 3.08

0.794 0.893 1.02 1.19 1.43 1.79

a. What can you conclude about the reactivities of the cysteinyl and histidyl residues of the protein? b. Which residue can you implicate in the active site? On what do you base the choice? Are the data conclusive concerning the assignment of a residue to the active site? Why or why not? c. After 1 h you dilute the enzyme incubated with iodoacetamide but no NADH. Do you expect the enzyme activity to be restored? Explain. 13. The initial velocity data shown in the table were obtained for an enzyme.
[S], (mM) 0.10 0.125 0.167 0.250 0.50 1.0 Velocity, (Ms-1) x 107 0.96 1.12 1.35 1.66 2.22 2.63

Each

assay

at

the

indicated

substrate

concentration was initiated by adding enzyme