1. What is the advantage of using a substrate concentration near the K m in I 50
ID: 1026767 • Letter: 1
Question
1. What is the advantage of using a substrate concentration near the Km in I50 determinations?
2. Estimate the I50 from the data below. How did you come to this conclusion?
[I] (mM) Activity (A/minute)
0 0.53
3.75 0.38
7.5 0.29
15 0.19
30 0.11
60 0.05
120 0.02
3. The data in the table below were obtained by performing Km determination experiments in the absence of inhibitor and in the presence of two different inhibitors.
a. What type of inhibitor is Inhibitor 1? Why?
b. What type of inhibitor is Inhibitor 2? Why?
c. What are the Ki values for Inhibitor 1 and Inhibitor 2?
Velocity (µmol/min)
Substrate (mM)
No Inhibitor
Inhibitor 1
(1 mM)
Inhibitor 2
(1 mM)
0.25
0.06
0.02
0.02
0.5
0.10
0.05
0.04
1
0.17
0.08
0.07
2
0.25
0.14
0.10
4
0.33
0.22
0.13
8
0.40
0.31
0.16
Velocity (µmol/min)
Substrate (mM)
No Inhibitor
Inhibitor 1
(1 mM)
Inhibitor 2
(1 mM)
0.25
0.06
0.02
0.02
0.5
0.10
0.05
0.04
1
0.17
0.08
0.07
2
0.25
0.14
0.10
4
0.33
0.22
0.13
8
0.40
0.31
0.16
Explanation / Answer
3)
a) and b) Inhibitors 1 & 2 both are competitive (reversible) type of inhibitors. It is because, in both the cases, velocity is increasing with increase in substrate concentration. However, in case of uncompetitive and noncompetitive type of inhibitors, the velocity always decreases.
c) The Ki values for the inhibitors are- Inhibitor 1= 3.118e+155 and Inhibitor 2= 1.365e+131. These are calculated using Graphpad software.
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