Inhibition of Bovine Cytochrome P‐450 11β by 18‐Unsaturated Progesterone Derivatives

The last step of aldosterone biosynthesis, an 11β‐hydroxylation followed by two 18‐hydroxylations, are catalyzed, in the bovine system, by the same enzyme, the cytochrome P ‐450 11β (deoxycorticosterone (DOC) → corticosterone → 18‐hydroxycorticosterone → aldosterone). The 11β‐ and 18‐hydroxylase activities were studied separately with a reconstituted enzymic system, using 11‐deoxy[ 14 C]corticosterone and [ 3 H]corticosterone, respectively, as substrates. The inhibition of 11β‐hydroxylase activity by corticosterone was competitive ( K i = 60 μM) showing that transformation of both substrates occurs at the same site. Double‐label/double‐substrate experiments, using an equimolar mixture of 11‐deoxy[ 14 C]corticosterone and [ 3 H]corticosterone, suggested that 18‐hydroxycorticosterone is directly formed from 11‐deoxycorticosterone without the intermediate corticosterone leaving the enzyme. Inhibitions by 18‐vinylprogesterone and 18‐ethynylprogesterone, potent inhibitors of aldosterone biosynthesis [Viger, A., Coustal, S., Pérard, S., Piffeteau, A. & Marquet, A. (1989) J. Steroid Biochem. 33 , 119–124], were characterized for both activities (11β‐ and 18‐hydroxylase). The value of reversible K i for the 18‐hydroxylation ( K i = 5 μM for 18‐vinylprogesterone and 30 μM for 18‐ethynylprogesterone) is lower than that for the 11β‐hydroxylation (30 μM and 100–150 μM, respectively); the former inhibitor is stronger than the latter for both steps. The binding of substrates and inhibitors to the active site was also examined by difference absorption spectroscopy. 18‐Vinylprogesterone gave rise to a type I spectrum with a K s value of 35 μM close to that of progesterone, while 18‐ethynylprogesterone showed a reverse type I spectrum with a much higher K s value (140 μM). Based on these results, a hypothetical model, involving a conformational change of the enzyme for the second step, is proposed.