Table 2

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).

3A26 I187T S368P/V369I2.24.614.43.4
3A26PpuMI I187T/S368P/V369I2.613.511.14.3

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.