Biochemistry I Fall Term

Enzyme Inhibition: Answer Sheet Example

Example Problem:
You are given a 4.0 mg/ml solution of an enzyme (Mr = 40,000 Da).
You determine initial velocities (vo) at several concentrations of substrate, [A], and at two concentrations of inhibitor, [I].
a) Determine Km and Vmax.
b) Determine KI for the inhibitor and the type of inhibition.
c) Submit your calculations and the graphs of 1/vo vs. 1/[A].
What we know from the problem:
1) Concentration of the protein = 4.0 mg/ml.
2) Protein Mr = 40,000 Da
What we found in the experiment:
  [ I ] = 0 [ I ] = 2 mM [ I ] = 5 mM
    [A] 1/[A]   vo 1/vo   vo 1/vo   vo 1/vo 
  0.25
  0.4
  0.7
  1.0
  2.0
  3.0
  5.0
  8.0
 12
 20

4.00
2.50
1.43
1.00
0.50
0.33
0.20
0.13
0.08
0.05

0.060
0.080
0.130
0.160
0.250
0.310
0.360
0.390
0.430
0.460

16.67
12.50
 7.69
 6.25
 4.00
 3.23
 2.78
 2.56
 2.33
 2.17

0.03
0.05
0.08
0.12
0.18
0.24
0.29
0.35
0.39
0.43

33.33
20.00
12.50
 8.33
 5.56
 4.17
 3.45
 2.86
 2.56
 2.33

0.02
0.03
0.06
0.08
0.13
0.18
0.24
0.31
0.35
0.39

50.00
33.33
16.67
12.50
 7.69
 5.56
 4.17
 3.23
 2.86
 2.56
[A] is mM; vo is mM/min. 1/[A] and the 1/vo values were calculated and plotted using the equation on the Enzyme Inhibition page in reciprocal form. (See Campbell's equation 5.18, p. 167).
 
Graph of the calculated results:

The Km = 2.0 mM.
The Vmax = 0.5 mM/min.
The KI = 3 mM.   Competitive inhibiton
Secondary Plots, shows how KI was determined from the slopes of the above lines.

EnzI.xls is the Excel spreadsheet used to calculate and graph these results, including the secondary plot; it can be copied to your disk.

Back to the Calculations & Graphing Index

8.20.03