Mechanism-based inactivation of bovine adrenal cytochromes P450 C-21 and P450 17 alpha by 17 beta-substituted steroids.

A series of progesterone derivatives has been studied as potential inactivators of the bovine adrenocortical cytochromes P450, P450 17 alpha, and P450 C-21. Replacement of the 21-methyl group of progesterone with a difluoromethyl group resulted in a selective inactivator of P450 C-21 in a reconstituted system. The loss of 21-hydroxylase activity caused by this compound exhibits a number of characteristics of mechanism-based inactivation including NADPH dependence, pseudo-first-order kinetics, saturability, irreversibility, and protection by substrate. In addition to the difluoro compound, 21,21-dichloroprogesterone, the acetylenic compound pregn-4-en-20-yn-3-one, and the olefinic compound pregna-4,20-dien-3-one all inactivate P450 C-21. In contrast, the only compound to inactivate the rabbit adrenal progesterone 21-hydroxylase is 21,21-dichloroprogesterone. In binding studies, the 21,21-dihalo steroids produce a greater maximal type I spectral shift of P450 C-21 than the two 17 beta-unsaturated steroids. The dihalo compounds inactivate P450 C-21 by both heme destruction and protein modification as shown by significant decreases in residual 21-hydroxylase activity and spectrally detectable P450 after incubation with P450 C-21 in a reconstituted system. Liquid chromatographic and mass spectral analyses of the organic extracts from these incubations showed that 21-pregnenoic acid is a major metabolite of the dihalo compounds with a partition ratio of 5 nmol of acid produced/nmol of P450 C-21 inactivated. This supports the hypothesis that inactivation proceeds in part through an acyl halide intermediate. In contrast, the acetylenic compound pregn-4-en-20-yn-3-one inactivates P450 C-21 mainly by protein modification, producing an NADPH-dependent irreversible type I spectral shift. The stoichiometry of inactivation is approximately 1.5 nmol of compound bound/nmol of enzyme inactivated, indicating selective modification of the enzyme at or near the substrate binding site.