Table 1. Summary of kinetic parameters for P450-BM3 and variants catalysing the Kemp elimination of 1.
| Catalyst* | Km (mM) | kcat (s−1) | kcat/Km(s−1 M−1) | kcat/Kuncat† |
|---|---|---|---|---|
| WT P450-BM3 | >6 | >1.5 | 240±60 | 1.3 × 106 |
| C400S | 2.7±0.4 | 3.8±0.4 | 1,400±150 | 3.3 × 106 |
| F87G | 2.1±0.4 | 2.7±0.3 | 1,300±140 | 2.3 × 106 |
| F87A | 2.0±0.3 | 1.2±0.1 | 600±50 | 1.0 × 106 |
| F87V | 0.50±0.04 | 1.1±0.1 | 2,200±200 | 0.9 × 106 |
| F87I | >4 | >1.5 | 320±40 | 1.3 × 106 |
| A82F | 0.27±0.03 | 8.4±0.4 | 31,000±1,500 | 7.0 × 106 |
| F87G/A82F | 1.3±0.2 | 11.5±0.7 | 8,800±700 | 10.0 × 106 |
| A82F/C400S | 0.61±0.13 | 2.9±0.4 | 4,800±700 | 2.5 × 106 |
*Assay conditions: 25 °C, 50 mM sodium phosphate buffer pH 8.0, 100 mM NaCl, 0.25 mM NADPH, 100 nM or 300 nM purified enzyme, 5% acetonitrile. Errors correspond to ±s.d. determined from at least three independent measurements.
†Rate accelerations for the Kemp eliminases were calculated based on the rate constant for the uncatalyzed reaction determined in ref. 10 (kuncat=1.16 × 10−6 s−1).