Allosteric Activation of Cytochrome P450 3A4 via Progesterone Bioconjugation.

Human cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of the majority of drugs. As such, it is implicated in many adverse drug-drug and food-drug interactions, and is of significant interest to the pharmaceutical industry. This enzyme is known to simultaneously bind multiple ligands and display atypical enzyme kinetics, suggestive of allostery and cooperativity. As well, evidence of a postulated peripheral allosteric binding site has provoked debate around its significance and location. We report the use of bioconjugation to study the significance of substrate binding at the proposed allosteric site and its effect on CYP3A4 activity. CYP3A4 mutants were created and covalently modified with various small molecules including progesterone. The labeled mutants displayed enhanced kinetic stability and improved activity in testosterone and 7-benzyloxy-(4-trifluoromethyl)coumarin oxidation assays. Our work applies a new strategy to study cytochrome P450 allostery and supports the hypothesis that substrate binding at the postulated allosteric site of CYP3A4 may induce functional cooperativity.