Mechanism-based inactivation of bovine cytochrome P-450(11beta) by 18-unsaturated progesterone derivatives.
Two 18-unsaturated progesterone derivatives, 18-vinylprogesterone (18-VP) and 18-ethynylprogesterone (18-EP) have proved to be potent inhibitors of the bovine cytochrome P-450(11beta), the enzyme involved in the last steps of aldosterone biosynthesis [Delorme, C., Piffeteau, A., Viger, A. & Marquet, A. (1995) Eur. J. Biochem. 232, 247-256]. In the present study, we demonstrate that these two compounds exhibit the characteristics of mechanism-based inactivators of this enzyme. Inactivation followed pseudo-first-order and saturation kinetics. The kinetic parameters of inactivation were k(i) = 0.11 min(-1) and Ki = 4 microM for 18-VP, and k(i) = 0.12 min(-1) and 22 microM for 18-EP. Inactivation of P-450(11beta) activity was strictly dependent on the presence of NADPH. Protection by the substrate deoxycorticosterone was observed, demonstrating a selective modification at the substrate-binding site. With radiolabeled 18-VP, inactivation was shown to be irreversible with a stoichiometry of 1.4 mol bound [3H]18-VP/mol inactivated cytochrome P-450(11beta). SDS/PAGE analysis of the [3H]18-VP-inactivated enzyme showed that, under conditions preventing heme dissociation, the P-450(11beta) band was labeled, while no labeling of the apoprotein was observed under denaturating conditions. Furthermore, the loss of catalytic activity could be correlated with the destruction of the P-450 chromophore evaluated by the FeII-CO versus FeII difference spectra. These arguments led us to propose that 18-vinylprogesterone inactivates cytochrome P-450(11beta) by heme destruction rather than by protein modification.
Delorme
C
C
Laboratoire de Chimie Organique Biologique, Université Pierre et Marie Curie, CNRS URA 493, Paris, France.
Piffeteau
A
A
Sobrio
F
F
Marquet
A
A
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006418:Heme / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D011374:Progesterone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D013252:Steroid 11-beta-Hydroxylase / Q000037:antagonists & inhibitors*; D013329:Structure-Activity Relationship
D004791:Enzyme Inhibitors; C069555:18-ethynylprogesterone; D006418:Heme; D011374:Progesterone; C069554:18-vinylprogesterone; D013252:Steroid 11-beta-Hydroxylase