Table 3.
Kinetic analysis of cytochrome P450 reductase, and the sW676A and sW6776H mutants
| Mutant | NADPH
|
NADH
|
NADPH/NADH A/B | ||||
|---|---|---|---|---|---|---|---|
| KM, μM | kcat, min−1 | kcat/KM(A), μM−1⋅min−1 | KM, μM | kcat, min−1 | kcat/KM(B), μM−1⋅min−1 | ||
| sCPR | 15.6 ± 7.1 | 4,082 ± 574 | 261.7 | 47,590.0 ± 12,570.0 | 2,677 ± 191 | 0.06 | 4362 |
| sW676H | 1.2 ± 0.3 | 453 ± 27 | 377.5 | 2,673.0 ± 644.7 | 1,897 ± 100 | 0.7 | 539 |
| sW676A | 0.2 ± 0.06 | 10 ± 0.5 | 50 | 318.2 ± 66.9 | 3,748 ± 159 | 11.8 | 4 |
Cytochrome c reduction has been measured with 5 pmol enzyme, with the exception of KMNADPH for sW677A, where 25 pmol was used. Kinetic analysis was performed with 50 μM cytochrome c and various concentrations of NADPH and NADH, as described in the legend to Table 1.