Mechanism‐Based Inactivation of Bovine Cytochrome P ‐450U 11β by 18‐Unsaturated Progesterone Derivatives

Two 18‐unsaturated progesterone derivatives, 18‐vinylprogesterone (18‐VP) and 18‐ethynylprogester‐ one (18‐EP) have proved to be potent inhibitors of the bovine cytochrome P ‐450 11β 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 K i = 4 μM for 18‐VP, and k i , = 0.12 min −1 and 22 μM for 18‐EP. Inactivation of P ‐450 11β 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 1.4mol bound [ 3 H]18‐VP/mol inactivated cytochrome P ‐450 11β SDS/PAGE analysis of the [ 3 H] 18‐VP–inactivated enzyme showed that, under conditions preventing heme dissociation, the P ‐450 11β 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 11β chromophore evaluated by the Fe II ‐CO versus Fe II difference spectra. These arguments led us to propose that 18‐vinylprogesterone inactivates cytochrome P ‐450 11β by heme destruction rather than by protein modification.