Chemistry 350 – Inhibitors Problem Set
1. The data below are for an enzyme inhibition study. Determine the Km, Vmax for
inhibited and uninhibited reactions. Determine the type of inhibition.
[S] µM
0.40
0.67
1.00
2.00
V (µmol/min)
with 0.0 nM
Inhibitor
0.22
0.29
0.32
0.40
V (µmol/min) V (µmol/min)
with 25 nM with 50 nM
Inhibitor
Inhibitor
0.21
0.26
0.30
0.36
0.20
0.24
0.28
0.32
2. The following data were obtained for a competitive inhibition study in which
the [I] = 3 uM for each determination of Vo in the presence of inhibitor.
Vmax = 200 uM P/min for both data sets.
a) Determine Km for the data obtained in the absence of inhibitor.
b) Determine Km, app for the data obtained in presence of inhibitor.
3. You have performed a series of experiments determining the Ki values for three
competitive inhibitors.
Ki (uM)
Inhibitor A Ki = 5uM, Inhibitor B Ki = 1uM, Inhibitor C Ki = 0.2uM
a) Which inhibitor binds with higher affinity to the free enzyme?
b) If the same concentration of inhibitor were used in each experiment, which
inhibitor would give the smallest value of Km,app?
c) If the value for Km is 1 uM, what is the ratio of Ki/Km for each inhibitor? How is
this related to the competing equilibria for binding of the substrate vs. the inhibitor
to the enzyme?
4. An enzyme is discovered to be implicated in a disease process. The enzyme is
known to have Km = 1.0 mM, and Vmax = 10.0 M/min for its natural substrate. As a
therapeutic strategy, a medicinal chemist tries to design a competitive inhibitor for
the enzyme. On testing, the inhibitor is shown to be a pure non-competitive
inhibitor with Vmax = 1.0 M/min at [I] = 1 mM. Assume [E]total is the same for all the
experiments.
a)Draw the double reciprocal plot for the enzyme in the presence and absence of the
inhibitor. Label which line on the plot corresponds to which experimental trial.
b) What is the inhibition constant, KI?
c) What would Km have been if the inhibitor had been competitive, assuming the
same KI?
5. Phenylketonuria is a genetically determined disease in which the enzyme
phenylalanine 4-hydroxylase is absent or has very low activity. The phenylalanine
hydroxylase activity of the liver was confined to a single protein of molecular weight
of approximately 108,000. It seems to consist of two polypeptide chains, each with
a molecular weight of approximately 54,000. By using the double- reciprocal plots,
the apparent Km values for phenylalanine were 3.5 x 10-4 M for the full-term infant
and 3.8 x 10-4 M for the adult preparations, respectively. For the synthetic cofactor,
apparent Km values were 6.8 x 10-5 M and 6.6 x 10-5 M, respectively . The activity of
the enzyme was assayed, by using various concentrations of phenylalanine, in the
presence and absence of 0.5 mM p-chlorophenylalanine . Iron-chelating and copper-chelating agents inhibited human phenylalanine hydroxylase. Thiol-binding
agents inhibited the enzyme but, as with the rat enzyme, phenylalanine both
stabilized the human enzyme and offered some protection against these inhibitors.
a. The Km value of the enzyme from human fetal liver for phenylalanine was
3.18 x 10-4 M. How does the affinity of the enzyme for the substrate change
with development?
b. What is the reaction catalyzed by phenylalanine 4-hydroxylase?
c. The apparent Km of the enzyme from human fetal liver for the synthetic
cofactor was 7.15 x 10-5 M. What changes with development can be inferred
from this information?
d. What kind of inhibitor would you guess they found p-chlorophenylalanine to
be? Why?
e. What does the next to the last sentence tell you?
f. What does the last sentence tell you?