Steroid derivatives as probes of adrenal cytochrome P-450 structure and function

Abstract

Cytochromes P450 metabolize lipophilic substrates to water-soluble products that are readily excreted from the body. The result of the action of hepatic P450 forms is generally detoxification, whereas P450s of the mammalian adrenal gland are responsible for steroid biosynthesis. To better understand the structure and function of two microsomal P450s of the adrenal cortex, P450 17α and P450 C-21, we have designed potential mechanism-based inactivators. These compounds bind reversibly to the enzyme before being metabolized to reactive intermediates that can then bind covalently to the P450, resulting in enzyme inactivation. Our hypothesis is that alteration of the substrate at the known site of enzyme attack may target the P450 for inactivation. Specifically, replacement of the progesterone 21-methyl group with a difluoromethyl group produced a selective inactivator of bovine adrenal P450 C-21. In contrast, the rabbit adrenal progesterone 21-hydroxylase is selectively inactivated by 21,21-dichloroprogesterone. Whether the substitution at the 17-carbon is a dihalomethyl-keto group, an olefinic group, or an acetylenic group, each compound binds reversibly to P450 C-21 as shown by a type I spectral shift. Inactivation of bovine adrenal P450 C-21 by 21,21-difluoroprogesterone is NADPH-dependent, follows pseudo first-order kinetics, and is virtually eliminated by the addition of the physiological substrate progesterone, thereby fulfilling the criteria for mechanism-based inactivation. Metabolism of the dihalo compounds to 21-pregnenoic acid suggests that an acyl halide intermediate is the chemical species responsible for enzyme inactivation. Both 21,21-dichloro and 21,21-difluoroprogesterone inactivate P450 C-21 by the destruction of P450 heme and by protein modification as evidenced by the loss of spectrally detectable P450 relative to the loss of enzyme activity. In contrast, 17β-ethynylprogesterone inactivates P450 C-21 mainly by protein modification and produces an NADPH-dependent, irreversible type I spectrum. Studies to isolate and identify an active site peptide of P450 C-21 were therefore undertaken using proteolytic digestion and high performance liquid chromatography. These 17β-substituted steroids proved useful as probes of P450 structure and function to obtain unique information about P450 oxidative potential, retention of substrate regioselectivity, catalytic efficiency, and the enzyme active site.