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# CHK 395G Kinetics Homework

1. The following experimental data were collected during a study of the catalytic activity of an
intestinal peptidase capable of hydrolyzing the dipeptide glycylglycine.

## Product formed Product formed

[S] (mM) (mol/min) [S] (mM) (mol/min)
1.5 0.21 4.0 0.33
2.0 0.24 8.0 0.40
3.0 0.28 16.0 0.45

From these data determine by graphical analysis (see Box 8-1) the values of Km and Vmax for this
enzyme preparation and substrate.

2. Many enzymes are inhibited irreversibly by heavy-metal ions such as Hg 2+, Cu2+, or Ag+, which can
react with essential sulfhydryl groups to form mercaptides:

## EnzSH + Ag+ EnzSAg + H+

The affinity of Ag+ for sulfhydryl groups is so great that Ag+ can be used to titrate SH groups
quantitatively. To 10 mL of a solution containing 1.0 mg/mL of a pure enzyme was added just
enough AgNo3 to completely inactivate the enzyme. A total of 0.342 mol of AgNO 3 was
required. Calculate the minimum molecular weight of the enzyme. Why does the value
obtained in this way give only the minimum molecular weight?

3. The enzymatic activity of lysozyme is optimal at pH 5.2 (see graph below). The active site of
lysozyme contains two amino acid residues essential for catalysis: Glu 35 and Asp52. The pKa
values of the carboxyl side chains of these two residues are 5.9 and 4.5, respectively. What is
the ionization state (protonated or deprotonated) of each residue at the pH optimum of
lysozyme? How can the ionization states of these two amino acid residues explain the pH-
activity profile of lysozyme shown below?

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4. Two different enzymes are able to catalyze the same reaction, A B. They both have the same
Vmax, but differ in their Km for the substrate A. For enzyme 1, the Km is 1.0 mM; for enzyme 2, the
Km is 10 mM. When enzyme 1 was incubated with 0.1 mM A, it was observed that B was
produced at a rate of 0.0020 mmoles/minute.

## a. What is the value of the Vmax of the enzymes?

b. What will be the rate of production of B when enzyme 2 is incubated with 0.1 mM A?
c. What will be the rate of production of B when enzyme 1 is incubated with 1M (i.e., 1000
mM) A?

5. An enzyme can catalyze a reaction with either of two substrates, S 1 or S2. The Km for S1 was
found to be 2.0 mM, and the Km for S2 was found to be 20 mM. A student determined that the
Vmax was the same for the two substrates. Unfortunately, he lost the page of his notebook and
needed to know the value of Vmax. He carried out two reactions: one with 0.1 mM S 1, the other
with 0.1 mM S2. Unfortunately, he forgot to label which reaction tube contained which
substrate. Determine the value of Vmax from the results he obtained:

1 0.5
2 4.8

## 6. An enzyme catalyzes a reaction at a velocity of 20 mol/min when the concentration of substrate

(S) is 0.01 M. The Km for this substrate is 1 x 10-5 M. Assuming that Michaelis-Menton kinetics
are followed, what will the reaction velocity be when the concentration of S is (a) 1 x 10 -5 M and
(b) 1 x 10-6 M?

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7. An enzyme catalyzes the reaction A B. The initial rate of the reaction was measured as a
function of the concentration of A. The following data were obtained:

0.05 0.08
0.1 0.16
0.5 0.79
1 1.6
5 7.3
10 13
50 40
100 53
500 73
1,000 76
5,000 79
10,000 80
20,000 80

## a. What is the Km of the enzyme for the substrate A?

b. What is the value of V0 when [A] = 43?
The above data was plotted as 1/V0 vs. 1/[A], and a straight line was obtained.
c. What is the value of the y-intercept of the line?
d. What is the value of the x-intercept of the line?

## 8. When 10 g of an enzyme of Mr 50,000 is added to a solution containing its substrate at a

concentration one hundred times the Km, it catalyzes the conversion of 75 mol of substrate into
product in 3 min. What is the enzymes turnover number?

9. It has been proposed that for enzymes that obey Michaelis-Menton kinetics, the Km value for an
enzyme may represent the approximate physiological substrate concentration in the cell.
Examine a typical plot of V versus [S] and explain why this appears to be a reasonable
suggestion.