| Abstrakt: |
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-45011β, 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 ki = 0.11 min-1 and Ki = 4 µM for 18-VP and ki =0.12 min-1 and 22 µM for 18-EP Inactivation of P-45011β 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 tool bound [3H]18-VP/mol inactivated cytochrome P-45011β SDS/PAGE analysis of the [3H]18-VP—inactivated enzyme showed that, under conditions preventing heme dissociation, the P-45011β 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 Fe11-CO versus Fe11 difference spectra. These arguments led us to propose that 18-vinylprogesterone inactivates cytochrome P-45011β by heme destruction rather than by protein modification. [ABSTRACT FROM AUTHOR] |
