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.