BIOCHEMISTRY 440
Problem Set #5
Lectures 12, 13, 14
* denotes problems that are most similar to possib le exam questions.
*1. Lactase is an enzyme found in the small intestines that catalyzes the hydrolysis of the milk sugar, lactose, to
yield glucose and galactose. Some individuals stop producing lactase as adults, making them lactose-intolerant.
Lactaid is an over-the-counter remedy for relief of lactose intolerance; the active ingredient is the enzyme
lactase. Use the following information to answer the questions. KM for lactose is 0.9 mM; Vmax is 3.5
µM/min/mg enzyme; lactose = 348 gm/mol; glucose = 182 gm/mol.
(a) Cow’s milk contains 4.8 mg lactose/100 mL milk. If you were to add lactase directly to a glass of
milk, how would [S] compare with KM?
(b) Will the enzyme added to milk work at its maximal velocity, half-maximal velocity, or another
velocity?
(c) Calculate the vo relative to Vmax.
(d) If you “pre-treat” milk with a Lactaid tablet that contains 200 mg of lactase, how long will it take for
the lactose concentration to be ½ the original concentration in milk? (Assume that steady-state kinetics will hold
throughout the time period.)
*2. You and your lab partner are studying the kinetics of an enzyme that can catalyze the reaction of either of
two substrates, S1 or S2. You are told that KM = 2.0 mM for S1 and KM = 20 mM for S2. Right at the end of lab
period, you determine that the Vmax is the same for both substrates. You need to catch a bus, so you ask your lab
partner to record the value in his notebook. Instead, he wrote the number on his hand and then washed his
hands before he could record it properly. You appeal to the good graces of the TA in charge of the lab, who
agrees to allow you (but NOT your hapless partner!) to go in and make another measurement. He supplies you
with two tubes, each containing 0.1 mM substrate (one with S1 & one with S2). Much to your chagrin, you
discover that the TA forgot to label the tubes. You decide to make the measurements anyway, hoping you’ll be
able to figure things out once you have the data. The results you obtain are:
Tube number Rate of formation of product (µmol/min)
1 0.5
2 4.8
Can you determine the value of Vmax with the information available to you? If so, what is it? If not, what other
information do you need?
*3. The enzyme, lysozyme, is abundant in egg white and in human tears. Lysozyme causes the lysis of bacteria
by catalyzing the hydrolytic cleavage of polysaccharides found in the cell walls. The enzymatic activity of
lysozyme is optimal at pH 5.2 and decreases at pH values above and below this optimum. The active site of
lysozyme contains two amino acid residues that are essential for catalysis: Glu-35 and Asp-52. The pKa values
for the two sidechains have been measured to be 5.9 and 4.5, respectively.
a. What is the predominant ionization state of each residue at the pH optimum for the enzyme?
b. Offer an hypothesis to explain the pH behavior of the enzyme on the basis of the ionization states for
these two active site residues.
c. Design three mutant proteins in which one, the other, or both residues are changed that will allow
you to test your hypothesis in part (b). Explain the specific issue each of your mutants will address and
a possible expected result for each of your designed mutants.
*4. Different enzymes that catalyze the same reaction are called “isozymes.” Often, different isozymes are
found in different tissues, where they catalyze the same reaction, but have different catalytic properties,
allowing them to work under different conditions. For example, there are several isozymes of the enzyme
called hexokinase, which catalyzes the first step in glycolysis, which you’ll be learning about in detail later this
quarter. The reaction catalyzed by hexokinase is:
glucose + ATP glucose-6-phosphate + ADP
