The Effects of a Naturally Occurring Genetic Polymorphism on the Catalytic Properties of Human Cytochrome P450 2B6.

Abstract

A reconstituted monooxygenase system containing purified P450 2B6 and NADPH-cytochrome P450-reductase (reductase), was used to investigate the catalytic properties of a naturally occurring mutation in P450 2B6 (2B6.4) in which a lysine is changed to an arginine. To probe this, several structurally unrelated mechanism-based inactivators of wild-type P450 2B6 were used. P450 2B6.4 was not inactivated by two of these compounds, 17-alpha-ethynylestradiol (17EE) and efavirenz. Subsequent studies were performed to determine which step(s) in the P450 catalytic cycle might be compromised in the mutant enzyme leading to the changes in the ability of the two compounds to act as mechanism-based inactivators. Studies on the binding of substrates and reductase to the P450s revealed similarities in the abilities of both enzymes to associate with substrates and the reductase. The reaction stoichiometries for the metabolism of efavirenz and 17EE indicated that the mutant enzyme was more uncoupled than the wild-type enzyme. However, the addition of cytochrome b5, a P450 redox partner, to the reconstitution mixture resulted in increased coupling of the P450 2B6.4-catalyzed reactions. In addition, in the presence of cytochrome b5, the mutant enzyme was now inactivated by both 17EE and efavirenz. These data lead us to suggest that one of the oxygen intermediates formed during the catalytic cycle, either the oxyferrous P450 and/or the iron-peroxo intermediate, may be less stable when formed by P450 2B6.4 compared to the same intermediates formed by the wild-type enzyme. In order to determine the site(s) of interaction between P450 2B6 and reductase, the cross-linking agent 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) was used to covalently link P450 2B6 to reductase. Cross-linked peptides were then identified by reconstituting the peptides in 18O-water following proteolysis, based on the principle that the cross-linked peptides would be expected to incorporate twice as many 18O atoms as non-cross-linked peptides. Subsequent mass spectrometric analyses of the resulting peptides lead to the identification of a cross-linked peptide candidate. De novo sequencing of the peptide suggests that it is a complex between the C-helix of the P450 and the connecting domain of reductase.