Steroid hydroxylase metabolite ratios of P-450s 3A12, the 3A26 I187I/S368P/V369I triple mutant, and the 3A26 triple mutant with 44 carboxy-terminal residues from 3A12 (construct L).
| P-450 | Testosterone | Progesterone | Androstenedione | |||
|---|---|---|---|---|---|---|
| 6β:2β | 6β:15β | 6β:16α | 6β:16β | |||
| 3A12 | 4.0 | 12.0 | 7.0 | 7.3 | ||
| 3A26 I187T S368P/V369I | 2.2 | 4.6 | 14.4 | 3.4 | ||
| 3A26PpuMI I187T/S368P/V369I | 2.6 | 13.5 | 11.1 | 4.3 | ||
Solubilized E. coli membrane preparations containing 10 pmol of wild-type 3A12, 3A26 I187T/S368P/V369I, or 3A26PpuMImutant were reconstituted with 40 pmol P-450 reductase and 10 pmol cytochrome b 5 and analyzed for progesterone, testosterone, and androstenedione hydroxylase activity. Substrate concentrations of 250 μM were used throughout. These ratios were derived from 2 to 4 incubations on two independent enzyme preparations.