MECHANISM-BASED INACTIVATION OF BOVINE CYTOCHROME P-450(11-BETA) BY 18-UNSATURATED PROGESTERONE DERIVATIVES

Two 18-unsaturated progesterone derivatives, 18-vinylprogesterone (18- VP) and 18-ethynylprogesterone (18-EP) have proved to be potent inhibi tors of the bovine cytochrome P-450(11 beta), the enzyme involved in t he last steps of aldosterone biosynthesis [Delorme, C., Piffeteau, A., Viger, A. & Marquet, A. (1995) fur. J. Biochem. 232, 247-256]. In the present study, we demonstrate that these two compounds exhibit the ch aracteristics of mechanism-based inactivators of this enzyme. Inactiva tion followed pseudo-first-order and saturation kinetics. The kinetic parameters of inactivation were k(i) = 0.11 min(-1) and K-i = 4 mu M f or 18-VP, and k(i) = 0.12 min(-1) and 22 mu M for 18-EP Inactivation o f P-450(11 beta) activity was strictly dependent on the presence of NA DPH. Protection by the substrate deoxycorticosterone was observed, dem onstrating a selective modification at the substrate-binding site. Wit h radiolabeled 18-VP, inactivation was shown to be irreversible with a stoichiometry of 1.4 mol bound [H-3]18-VP/mol inactivated cytochrome P-450(11 beta). SDS/PAGE analysis of the [H-3]18-VP-inactivated enzyme showed that, under conditions preventing heme dissociation, the P-450 (11 beta) band was labeled, while no labeling of the apoprotein was ob served under denaturating conditions. Furthermore, the loss of catalyt ic activity could be correlated with the destruction of the P-450 chro mophore evaluated by the Fe-II-CO versus Fe-II difference spectra. The se arguments led us to propose that 18-vinylprogesterone inactivates c ytochrome P-450(11 beta) by heme destruction rather than by protein mo dification.