Table 1

Steroid hydroxylase activities of P-450 3A12 and 3A26 double and triple mutants

P-450ProgesteroneTestosteroneAndrostenedione
16α15β16β
3A125.8, 6.60.84, 0.910.21, 0.302.6, 3.50.63, 0.914.4, 4.50.53, 0.60
3A26 S368P/V369I0.70, 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/V369I2.1, 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.