Allosteric Activation of Cytochrome P450 3A4 via Progesterone Bioconjugation

Human cytochrome P450 3A4 (CYP3A4) is responsible for themetabolism 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 pharmaceuticalindustry. This enzyme is known to simultaneously bind multiple ligands and displayatypical enzyme kinetics, suggestive of allostery and cooperativity. As well, evidence of apostulated peripheral allosteric binding site has provoked debate around its significanceand location. We report the use of bioconjugation to study the significance of substratebinding at the proposed allosteric site and its effect on CYP3A4 activity. CYP3A4mutants were created and covalently modified with various small molecules includingprogesterone. The labeled mutants displayed enhanced kinetic stability and improvedactivity in testosterone and 7-benzyloxy-(4-trifluoromethyl)coumarin oxidation assays.Our work applies a new strategy to study cytochrome P450 allostery and supports thehypothesis that substrate binding at the postulated allosteric site of CYP3A4 may inducefunctional cooperativity