Steroid hydroxylase activities of P-450 3A12 and 3A26 double and triple mutants
| P-450 | Progesterone | Testosterone | Androstenedione | ||||
|---|---|---|---|---|---|---|---|
| 6β | 16α | 15β | 6β | 2β | 6β | 16β | |
| 3A12 | 5.8, 6.6 | 0.84, 0.91 | 0.21, 0.30 | 2.6, 3.5 | 0.63, 0.91 | 4.4, 4.5 | 0.53, 0.60 |
| 3A26 S368P/V369I | 0.70, 0.98 (14) | 0.14, 0.14 (16) | 0.22, 0.26 (94) | 1.4, 1.6 (49) | 0.53, 0.63 (75) | 1.6, 2.7 (48) | 0.30, 0.47 (73) |
| 3A26 I187T S368P/V369I | 2.1, 2.4 (36) | 0.16, 0.15 (18) | 0.69, 0.62 (257) | 3.4, 2.7 (100) | 1.7, 1.1 (182) | 3.7, 5.8 (107) | 1.1, 1.6 (239) |
Solubilized E. coli membrane preparations containing 10 pmol of 3A12, 3A26 S368P/V369I, or 3A26 I187T/S368P/V369I mutants were reconstituted with 40 pmol P-450 reductase and 10 pmol cytochromeb 5 and analyzed for progesterone, testosterone, and androstenedione hydroxylase activity. Substrate concentrations of 250 μM were used throughout. Rates are given in nanomoles product per minute per nanomole P-450 and represent mean of 2 to 4 incubations with two separate preparations of each enzyme. Numbers in parentheses represent 3A26 mutant activity as a percentage of 3A12 activity.