Table 4.
Kinetic analyses of the active site mutants
| Substrate/cofactor | Carboxylase | Km, μM | Vmax, pmol/min | Vmax/Km, pmol/min per μM × 102 |
|---|---|---|---|---|
| EEL | Wild type | 200 | 545 | 273 |
| C99S | 1,600 | 5 | <1 | |
| C450S | 1,100 | 38 | 3 | |
| KH2 | Wild type | 29 | 493 | 1,700 |
| C99S | 4 | 5 | 125 | |
| C450S | 5 | 40 | 800 | |
| CO2 | Wild type | 900 | 513 | 57 |
| C99S | 1,000 | 5 | <1 | |
| C450S | 1,100 | 38 | 3 |
Lysates were used instead of microsomes because the higher activities obtained for the mutants made these assays possible. A total of 100 μg of lysate was assayed, and the carboxylase levels, determined by quantitative Western analysis, were normalized to the wild-type levels. Each value was determined three times (supplemental Methods), and the error for each experiment was +/− 5%.