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

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.

7. What is the steady-state approximation and

under what conditions is it valid?

8. Assume that an enzyme-catalyzed reaction

follows Michaelis-Menten kinetics with KM of

1µM. The initial velocity is 0.1µM/min at 10mM

substrate. Calculate the initial velocity at 1mM,

10µM and 1µM 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:

E + S ES 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 20µM.

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.

Product

formed

Time

k1

k2

k3

k4

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) (NADH Present)

Time

(min)

Activity

(U/mg)

His Cys Time

(min)

Activity

(U/mg)

His Cys

0 1000 20 12 0 1000 20 12

15 560 18.2 11.4 15 975 20 11.4

30 320 17.3 10.8 30 950 20 10.8

45 180 16.7 10.4 45 925 19.8 10.4

60 100 16.4 10.0 60 900 19.6 10.0

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) Velocity, (Ms-1) x 107

0.10 0.96

0.125 1.12

0.167 1.35

0.250 1.66

0.50 2.22

1.0 2.63

Each assay at the indicated substrate

concentration was initiated by adding enzyme

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 10µM. The data are shown in the

table.

Velocity Velocity Velocity

[S] (Ms-1) x 107 (Ms-1) x 107 (Ms-1) x 107

(mM) Uninhibited Inhibited A Inhibited B

0.333 1.65 1.05 0.794

0.4 1.86 1.21 0.893

0.5 2.13 1.43 1.02

0.666 2.49 1.74 1.19

1.0 2.99 2.22 1.43

2.0 3.72 3.08 1.79