Inhibition of bovine cytochrome P-450(11 beta) by 18-unsaturated progesterone derivatives.
The last step of aldosterone biosynthesis, an 11 beta-hydroxylation followed by two 18-hydroxylations, are catalyzed, in the bovine system, by the same enzyme, the cytochrome P-450(11 beta) (deoxycorticosterone (DOC)-->corticosterone-->18-hydroxycorticosterone-->aldosterone). The 11 beta- and 18-hydroxylase activities were studied separately with a reconstituted enzymic system, using 11-deoxy[14C]corticosterone and [3H]corticosterone, respectively, as substrates. The inhibition of 11 beta-hydroxylase activity by corticosterone was competitive (Ki = 60 microM) showing that transformation of both substrates occurs at the same site. Double-label/double-substrate experiments, using an equimolar mixture of 11-deoxy[14C]corticosterone and [3H]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 (11 beta- and 18-hydroxylase). The value of reversible Ki for the 18-hydroxylation (Ki = 5 microM for 18-vinylprogesterone and 30 microM for 18-ethynylprogesterone) is lower than that for the 11 beta-hydroxylation (30 microM and 100-150 microM, 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 Ks value of 35 microM close to that of progesterone, while 18-ethynylprogesterone showed a reverse type I spectrum with a much higher Ks value (140 microM). Based on these results, a hypothetical model, involving a conformational change of the enzyme for the second step, is proposed.
Delorme
C
C
Laboratoire de Chimie Organique Biologique, Université Pierre et Marie Curie, URA CNRS 493, Paris, France.
Piffeteau
A
A
Viger
A
A
Marquet
A
A
D000818:Animals; D002417:Cattle; D019405:Cytochrome P-450 CYP11B2; D065607:Cytochrome P-450 Enzyme Inhibitors; D004794:Enzyme Repression; D007700:Kinetics; D008024:Ligands; D011374:Progesterone / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D013252:Steroid 11-beta-Hydroxylase / Q000037:antagonists & inhibitors*
D065607:Cytochrome P-450 Enzyme Inhibitors; D008024:Ligands; D011374:Progesterone; D019405:Cytochrome P-450 CYP11B2; D013252:Steroid 11-beta-Hydroxylase