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 (6 beta-hydroxyprogesterone and 16 alpha-hydroxyprogesterone), two secondary metabolites (6 beta,16 alpha-dihydroxyprogesterone and 6-ketoprogesterone), and one tertiary metabolite (6-keto-16 alpha-hydroxyprogesterone). The Km,app for the formation of these products from progesterone was determined to be approximately 0.5 microM, while the Km,app for metabolism of 6 beta- and 16 alpha-hydroxyprogesterone was found to be 5-10 microM. The ratio of primary to secondary metabolites did not change significantly at progesterone concentrations from 6 to 150 microM, 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 6 beta,16 alpha-dihydroxyprogesterone and 6-keto-16 alpha-hydroxyprogesterone was observed in incubations containing a mixture of radiolabeled progesterone and unlabeled 6 beta-hydroxyprogesterone or 16 alpha-hydroxyprogesterone. Incubation of 6 beta-hydroxyprogesterone with a reconstituted system in an atmosphere of 18O2 resulted in greater than 90% incorporation of 18O in the 16 alpha-position of 6 beta,16 alpha-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.