CHM 332: Fall 2020

CHM 332: Fall 2020

Homework #4

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

capable of hydrolyzing the dipeptide glycylglycine.

 

Glycylglycine + H2O → 2 glycine

 

 

[S] (mM)

Product formed

(µmol/min)

 

[S] (mM)

Product formed

(µmol/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 the values of Km and Vmax for this enzyme preparation and

substrate.

 

2. Many enzymes are inhibited irreversibly by heavy-metal ions such as Hg2+, Cu2+, or Ag+, which can react with essential sulfhydryl groups to form mercaptides:

 

Enz−SH + Ag+ → Enz−S−Ag + 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 0.432 µmol of AgNO3 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: Glu35

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 at right?

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, S1 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 S1, 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:

 

Tube Number Rate of formation of product

1 0.5

2 4.8