Enzyme Inhibition Problems

1. 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.

200
No Inhibitor
180
160
140
Vo (uM P/min)

+ Inhibitor
120
100
80
60
40
20
0
0 50 100 150 200
[Substrate] (uM)

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.
c) Using the equation in the lecture notes, calculate the value for Ki.

2. You have performed a series of experiments determining the Ki values for

three competitive inhibitors. The following table lists the results:

Inhibitor Ki (uM)

A 5
B 1
C 0.2

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?
 Enzyme Inhibition Answers

1. 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.

200
180
160
140
Vo (uM P/min)

120
100
Km = 10 uM
80
Km,app = 30 uM
60
40
20
0
0 50 100 150 200
[Substrate] (uM)

Vmax = 200 uM P/min for both data sets.

a) Determine Km for the data obtained in the absence of inhibitor. Since Vmax
= 200, Vmax/2 = 100. Km = [S] that gives Vo = Vmax/2, so Km = 10 uM. .
b) Determine Km, app for the data obtained in presence of inhibitor. Km, app is
determined the same way, so Km, app = 30 uM.
c) Using the equation in the lecture notes, calculate the value for Ki.

Km, app = Km (1+ [I]/Ki)

30 uM = 10 uM(1 + 3 uM/Ki )
3 = 1 + (3 uM/Ki)
2 = 3 uM/Ki

Ki = 3 uM/2 = 1.5 uM
2. You have performed a series of experiments determining the Ki values for
three competitive inhibitors. The following table lists the results:

Inhibitor Ki (uM)

A 5
B 1
C 0.2

a) Which inhibitor binds with higher affinity to the free enzyme? The inhibitor with
the lowest Ki (a dissociation constant) binds with the highest affinity, so inhibitor
C.
b) If the same concentration of inhibitor were used in each experiment, which
inhibitor would give the smallest value of Km,app? The smallest Km,app value
implies the weakest binding of I, or the least amount of competition for E.
Inhibitor A would give the lowest value for 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?

The ratio of Ki/Km directly compares the binding constants for both the inhibitor
and the substrate.
If Ki/Km > 1, the substrate is bound tighter, so inhibitor will not have that much
effect on kinetics.
If Ki/Km = 1, both substrate and inhibitor bind with same affinity.
If Ki/Km < 1, the inhibitor binds with greater affinity, therefore will have a
stronger effect on the kinetics.

In this case, Ki/Km = 5, 1, and 0.2 for A, B, and C, respectively. Thus, as was the
case in (a), C binds with greatest affinity and will have a greater effect on the
enzyme kinetics than A or B.