INHIBITION OF BOVINE CYTOCHROME P-450(11-BETA) BY 18-UNSATURATED PROGESTERONE DERIVATIVES

The last step of aldosterone biosynthesis, an 11 beta-hydroxylation fo llowed by two 18-hydroxylations, are catalyzed, in the bovine system, by the same enzyme, the cytochrome P-450(11 beta) (deoxycorticosterone (DOG) --> corticosterone --> 18-hydroxycorticosterone --> aldosterone ). The 11 beta- and 18-hydroxylase activities were studied separately with a reconstituted enzymic system, using 11-deoxy[C-14]corticosteron e and [H-3]corticosterone, respectively, as substrates. The inhibition of 11 beta-hydroxylase activity by corticosterone was competitive (K- i = 60 mu M) showing that transformation of both substrates occurs at the same site. Double-label/double-substrate experiments, using an equ imolar mixture of 11-deoxy[C-14]corticosterone and [H-3]corticosterone , suggested that 18-hydroxycorticosterone is directly formed from 11-d eoxycorticosterone without the intermediate corticosterone leaving the enzyme. Inhibitions by 18-vinylprogesterone and 18-ethynylprogesteron e, potent inhibitors of aldosterone biosynthesis [Viger, A., Coustal, S., Perard, S., Piffeteau, A. and Marquet, A. (1989) J. Steroid Bioche m. 33, 119-124], were characterized for both activities (11 beta- and 18-hydroxylase). The value of reversible K-i for the 18-hydroxylation (K-i = 5 mu M for 18-vinylprogesterone and 30 mu M for 18-ethynylproge sterone) is lower than that for the 11 beta-hydroxylation (30 mu M and 100-150 mu M, respectively); the former inhibitor is stronger than th e latter for both steps. The binding of substrates and inhibitors to t he 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 mu M close to that of progesterone, while 18-ethynylprogesteron e showed a reverse type I spectrum with a much higher K-s value (140 m u M). Based on these results, a hypothetical model, involving a confor mational change of the enzyme for the second step, is proposed.