Cytochrome P-450 scc-catalyzed production of progesterone from 22 R-hydroxycholest-4-en-3-one by way of 20,22-dihydroxycholest-4-en-3-one

Abstract

Transient accumulation of a dihydroxylated steroid was found when 22 R-hydroxycholest-4-en-3-one was used as the substrate for a reconstituted cholesterol side-chain cleavage system derived from bovine adrenocortical mitochondria. The indications were that the accumulated steroid was an intermediate in the cytochrome P-450 scc-catalyzed reaction. The retention time of the accumulated intermediate was identical with that of authentic 20,22-dihydroxycholest-4-en-3-one on HPLC. When 22 R-hydroxycholesterol and 22 R-hydroxycholest-4-en-3-one were incubated simultaneously, the total amount of reaction products was essentially the same as that observed with 22 R-hydroxycholest-4-en-3-one alone. Under the conditions employed, the apparent turnover number of cytochrome P-450 scc for 22 R-hydrocholesterol was calculated to be 77 nmol/min/nmol P-450 from the amount of pregnenolone formed, whereas the apparent turnover number for 22 R-hydroxycholest-4-en-3-one was 64 nmol/min/nmol P-450 with respect to the intermediate formation and 77 nmol/min/nmol P-450 with respect to the progesterone formation. The apparent turnover number for 20,22-dihydroxycholest-4-en-3-one was about 125 nmol/min/nmol P-450, which was not significantly different from that of 20,22-dihydroxycholesterol. The apparent K m for 22 R-hydroxycholesterol was about 20 μM and those for 22 R-hydroxycholest-4-en-3-one and 20,22-dihydroxycholest-4-0en-3-one were 50 and 40 μM, respectively. Thus, 22 R-hydroxycholest-4-en-3-one was efficiently metabolized to progesterone by way of 20,22-dihydroxycholest-4-en-3-one by cytochrome P-450 scc.