Problems (Enzyme Kinetics)

Estimation of Vmax and KM by Inspection of the Graph in the Absence of Inhibitor and
Presence of Inhibitor

1. An experiment measuring velocity versus substrate concentration was run, first in the
absence of inhibitor X, and then in the presence of inhibitor X. The following data were
obtained:

[S], μM Velocity in absence of X, Velocity in presence of X,

μmol/min μmol/min

2.0 2.22 1.66

2.5 2.66 2.0
3.3 3.3 2.5
5.0 4.4 3.33
10.0 6.5 5.0

For this experiment,

i) calculate the Km and Vmax both in the absence and presence of inhibitor X.
ii) If substance X is an inhibitor, What kind of inhibition it is?

Answers: a) Take the reciprocals of both [S], and V; Determine 1/[S], 1/ V the absence of
Inhibitor (NoI), 1/VnoI, and in the Presence of Inhibitor (I), 1/VI

[S] 1/[S] VnoI 1/VnoI VI 1/VI

2.0 0.5 2.22 0.45 1.66 0.6

2.5 0.4 2.66 0.37 2.0 0.5
3.3 0.3 3.3 0.3 2.5 0.4
5.0 0.2 4.4 0.225 3.33 0.3
10.0 0.1 6.5 0.15 5.0 0.2

b) Plot 1/[S] vs. 1/VnoI and 1/[S] vs. 1/VI on same graph.

• From the cross-point on Y axis and X axis, find out the Km and Vmax value for
both the absence of Inhibitor (NoI), 1/VnoI, and in the Presence of Inhibitor (I), 1/VI
• From the result and pattern of the curve, interpret about the inhibition.
 • From the Y intercept and X intercept for NoI,
1/VmaxNoI = 0.072 Therefore, Vmax NoI = 1/0.072 = 13.89
– 1/KmNoI = – 0.1 Therefore, Km NoI = 1/0.1 = 10
• From the Y intercept and X intercept for I,
1/VmaxI = 0.1 Therefore, VmaxI = 1/0.1 = 10
– 1/KmI = – 0.1 Therefore, KmI = 1/0.1 = 10

By observing results and pattern of the graph, it was found that, Km values for in the absence and
presence of Inhibitor was unchanged i.e. 10. On the other hand, Vmax value was found to be
decreased in the presence of Inhibitor, i.e. decreased from 13.89 to 10 umole/min.
It can be inferred that the inhibition was non-competitive.
2. An experiment measuring velocity versus substrate concentration was run, first in the
absence of inhibitor X, and then in the presence of inhibitor X. The following data were
obtained:

[S], μM Velocity in absence of X, Velocity in presence of X,

μmol/min μmol/min

2.0 2.33 1.66

2.5 2.78 2.0
3.3 3.4 2.5
5.0 4.34 3.33
10.0 6.0 5.0

For this experiment,

i) calculate the Km and Vmax both in the absence and presence of inhibitor X.
ii) If substance X is an inhibitor, What kind of inhibition it is?

Answers: a) Take the reciprocals of both [S], and V; Determine 1/[S], 1/ V the absence of
Inhibitor (NoI), 1/VnoI, and in the Presence of Inhibitor (I), 1/VI

[S] 1/[S] VnoI 1/VnoI VI 1/VI

2.0 0.5 2.33 0.43 1.66 0.6

2.5 0.4 2.78 0.36 2.0 0.5
3.3 0.3 3.4 0.295 2.5 0.4
5.0 0.2 4.34 0.23 3.33 0.3
10.0 0.1 6.0 0.165 5.0 0.2

b) Plot 1/[S] vs. 1/VnoI and 1/[S] vs. 1/VI on same graph.

• From the cross-point on Y axis and X axis, find out the Km and Vmax value for
both the absence of Inhibitor (NoI), 1/VnoI, and in the Presence of Inhibitor (I), 1/VI
• From the result and pattern of the curve, interpret about the inhibition.
 • From the Y intercept and X intercept for NoI,
1/VmaxNoI = 0.1 Therefore, Vmax NoI = 1/0.1 = 10
– 1/KmNoI = – 0.1 Therefore, Km NoI = 1/0.1 = 10
• From the Y intercept and X intercept for I,
1/VmaxI = 0.1 Therefore, VmaxI = 1/0.1 = 10
– 1/KmI = – 0.15 Therefore, KmI = 1/0.15 = 15

By observing results and pattern of the graph, it was found that, Km values for the presence of
Inhibitor was found increased i.e. 15 than in the absence i.e. 10. On the other hand, Vmax value
was found to be was found to be unchanged i.e. in both cases it was 10 umole/min.
Therefore, it can be inferred that the inhibition was competitive.