TABLE 2.
Apparent kinetic parameters (50 mm Tris, pH 7.4, with and without the addition of 200 mm KCl) for reduction of Fdx by FdR using cytochrome c as the terminal electron acceptor, and NADH turnover by the P450 enzymes with different substrates for which the first electron transfer from Arx to the P450 is the slow step
| kcat | Km | ||
|---|---|---|---|
| s−1 | μm | ||
| FdR/Fdx/cyt c | |||
| ArR/Arx | 280 ± 12 | 2.9 ± 0.41 | |
| ArR/Arx, KCl | 320 ± 29 | 15 ± 2.5 | |
| PdR/Arx | –a | –a | |
| PuR/Arx | 250 ± 15 | 17 ± 2.2 | |
| ArR/Pdx | 37 ± 5.0 | 70 ± 14.6 | |
| PuR/Pdx | 600 ± 10 | 12 ± 0.5 | |
| PdR/Pdx | 410 ± 9.0 | 23 ± 1.0 | |
| ArR/Pux | 66 ± 1.2 | 0.4 ± 0.06 | |
| PdR/Pux | 160 ± 10 | 29 ± 3.4 | |
| PuR/Pux | 260 ± 6 | 4 ± 0.3 | |
| ArR/Arx/P450 | |||
| CYP101B1 β-ionone | 68 ± 1.9 | 11 ± 0.74 | |
| CYP101B1 β-ionone. KCl | 83 ± 8.8 | 160 ± 22 | |
| CYP101C1 β-iononeb | 48 ± 0.8 | 0.93 ± 0.04 | n = 1.9 ± 0.1 |
| CYP101C1 β-ionone, KClb | 46 ± 1.8 | 16 ± 1.1 | n = 1.6 ± 0.1 |
| CYP101D1 camphor | 41 ± 0.6 | 2.9 ± 0.14 | |
| CYP101D1 camphor, KCl | 31 ± 0.8 | 30 ± 1.5 | |
| CYP101D2 camphor | 39 ± 0.8 | 1.7 ± 0.12 | |
| CYP101D2 camphor, KCl | 30 ± 0.5 | 9.0 ± 0.38 | |
| CYP111A2 linalool | 91 ± 2.7 | 3.7 ± 0.31 | |
| CYP111A2 linalool, KCl | 63 ± 8.8 | 130 ± 24 | |
a When PdR was used with Arx the rates of reduction of cytochrome c was very slow. When 40 μm Arx was used the turnover rate was less than 20 s−1 and the system did not show saturation suggesting the kcat is low and the Km is high for PdR/Arx, which agree with data observed previously (supplemental Table S1) (20).
b All data showed good fits to hyperbolic behavior with the exception of CYP101C1 with β-ionone, which was fitted to the Hill equation.