Evidence for concerted kinetic oxidation of progesterone by purified rat hepatic cytochrome P-450g

Purified cytochrome P-450g, a male-specific rat hepatic isozyme, was observed to metabolize progesterone to two primary metabolites (6β-hydroxyprogesterone and 16α-hydroxyprogesterone), two secondary metabolites (6β,16α-dihydroxyprogesterone and 6-ketoprogesterone), and one tertiary metabolite (6-keto-16α-hydroxyprogesterone). The K/sub m,app/ for the formation of these products from progesterone was determined to be approximately 0.5 μM, while the K/sub m,app/ for metabolism of 6β- and 16α-hydroxyprogesterone was found to be 5-10 μM. The ratio of primary to secondary metabolites did not change significantly at progesterone concentrations from 6 to 150 μM, and a lag in formation of secondary metabolites was not observed in 1-min incubations. Concerted oxidation of progesterone to secondary products without the intermediate products leaving the active site was suggested by these results and confirmed by isotopic dilution experiments in which little or no dilution of metabolically formed 6β,16α-dihydroxyprogesterone and 6-keto-16α-hydroxyprogesterone was observed in incubations containing a mixture of radiolabeled progesterone and unlabeled 6β-hydroxyprogesterone or 16α-hydroxyprogesterone. Incubation of 6β-hydroxyprogesterone with a reconstituted system in an atmosphere of 18I2 resulted in > 90% incorporation of 18O in the 16α-position of 6β,16α-dihydroxyprogesterone but no incorporation of 18O into 6-ketoprogesterone, even though the reaction was dependent upon enzyme and O2, and not inhibited by mannitol, catalase, or superoxide dismutase. Factors which characterize the metabolism of progesterone by cytochrome P-450g in terms of active-site constraints and the catalytic competence of the enzyme in microsomes were also explored