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Biochemistry 410

BioTutorial 253

Biochemistry

Enzyme Kinetics: 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].
a) Determine Km and Vmax.
b) Calculate kcat for the reaction.
c) Submit your calculations and a graph of 1/vo vs. 1/[A].
What we know from the problem:
1) Concentration of the enzyme = 4.0 mg/ml.
2) Enzyme Mr = 40,000 Da
What we found in the experiment:
    [A]
  (mM)		     vo
(mM/min)		   1/[A]
(mM-1)		   1/vo
(min/mM)		 
  0.25
  0.4
  0.7
  1.0
  2.0
  3.0
  5.0
  8.0
 12
 20

0.060
0.080
0.130
0.160
0.250
0.310
0.360
0.390
0.430
0.460

4.00
2.50
1.43
1.00
0.50
0.33
0.20
0.13
0.08
0.05

16.67
12.50
 7.69
 6.25
 4.00
 3.23
 2.78
 2.56
 2.33
 2.17
The enzyme concentration = (4 mg/l)/(40,000 g/mol) = 0.10mM (10-7 M).
The last two columns were calculated using equations from the Enzyme Kinetics page.
 
Graph of the calculated results:

The Km = 2.0 mM; Vmax = 0.5 mM/min.
The kcat = Vmax/[enzyme] = (0.5 mM/min)/(0.10mM) = 5¥103 min-1.

The Excel spreadsheet is used to calculate and graph these results; it can be copied to your disk.

Calculations & Graphing Index

10.2.00

 

 

 

 

 

 

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March 21, 2001

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