A covalently attached progesterone molecule outcompetes the binding of free progesterone at an allosteric site of cytochrome P450 3A4.

Because of its exceptional substrate promiscuity, human P450 3A4 (CYP3A4) is arguably the most important drug-metabolizing enzyme. CYP3A4 also has the particularity of binding multiple ligands simultaneously, which is associated with heterotropic or homotropic, positive or negative, cooperativity or allostery. Solving the kinetics of such complex systems remains challenging, and so is identifying the binding pockets involved. Progesterone (PRG) is a known allosteric activator of CYP3A4-catalyzed 7-benzyloxy-4-trifluoromethylcoumarin (BFC) debenzylation. We report herein the use of bioconjugation as a successful strategy to identify this PRG allosteric site. A progesterone analogue (PGM) was covalently attached, separately at several locations, near a peripheral binding pocket previously proposed to be an allosteric site. Studies of BFC debenzylation in the presence of free PRG revealed that two of the bioconjugates successfully positioned the covalently attached PGM moiety in a way that mimcs the allosteric activation observed with free PRG. Interestingly, the PGM bioconjugate with the better fit yielded a higher permanent activation of the enzyme.