Question

You have an enzyme that uses two substrates in a random reaction (the order of binding of the substrates is random). And the reaction A + B à P is catalyzed by the enzyme bringtogetherase

You have carried out five sets of reaction rates Keeping B constant and varying A and then increasing B and Vary ing A again until all combinations of A and B are measured. In the reaction you used 0.02 mg of bringtogetherase which is a 120,000 Dalton protein.

The set of data is as follows

[A] mM             Vo nmoles per min                [B] mM

0.05                 363.64                                     0.5

0.03333           322.58                                     0.5

0.0200             263.16                                     0.5

0.0125             208.33                                     0.5

0.01                 181.81                                     0.5

0.0050             625                                          1

0.03333           555.56                                     1

0.0200             454.54                                     1

0.0125             357.14                                     1

0.01                 312.5                                       1

0.05                 952.38                                     2

0.03333           847.45                                     2

0.0200             714.29                                     2

0.0125             555.56                                     2

0.01                 487.80                                     2

0.05                 1250                                        5

0.03333           1111.11                                   5

0.0200             909.1                                       5

0.0125             724.64                                     5

0.01                 632.91                                     5

0.05                 1515.15                                   10

0.03333           1351.35                                   10

0.0200             1086.96                                   10

0.0125             847.46                                     10

0.01                 740.74                                     10

Determine the Km for A, the Km for B the Vmax and the turnover number for the enzyme.

To get the Vmax you need to replot the intercepts [Vmax apparent] vs the other substrate.

Answer 1

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