Induced Fit Describes Ligand Binding to Membrane-Associated Cytochrome P450 3A4

Abstract

Cytochrome P450 3A4 (CYP3A4) is the dominant P450 involved in human xenobiotic metabolism. Competition for CYP3A4 therefore underlies several adverse drug-drug interactions. Despite its clinical significance, the mechanisms CYP3A4 uses to bind diverse ligands remain poorly understood. Highly monodisperse CYP3A4 embedded in anionic lipoprotein nanodiscs containing an equal mixture of POPC and POPG were used to determine which of the limiting kinetic schemes that include protein conformational change, conformational selection (CS) or induced fit (IF), best described the binding of four known irreversible inhibitors. Azamulin, retapamulin, pleuromutilin, and mibrefadil binding to CYP3A4 NDs conformed to a single-site binding model. Exponential fits of stopped-flow UVvis absorption spectroscopy data supported multiple-step binding mechanisms. Trends in the rates of relaxation to equilibrium with increasing ligand concentrations were ambiguous to whether IF or CS was involved; however, global fitting and consideration of the rate constants favored an IF mechanism. In the case of mibrefadil, a transient complex was observed in the stopped-flow UV-vis experiment, definitively assigning the presence of IF in ligand binding.While these studies only consider a small region of CYP3A4's vast ligand space, they provide kinetic evidence that CYP3A4 can utilize an IF mechanism.

Kinetics of Intermediate Release Enhances P450 11B2-Catalyzed Aldosterone Synthesis

“…For all conditions, the k obs of the “fast” phase increased linearly with increasing ligand concentration (Figure , I–L), while the k obs trends for the second phase were variable (Figure , M–P). These results suggest that ligand binding to P450 11B enzymes might involve either conformational selection or induced fit mechanisms, analogous to what has been reported with other cytochrome P450 enzymes. − …”

“…Several mammalian cytochrome P450 enzymes that catalyze sequential reactions, particularly cytochromes P450 19A1 and P450 17A1, demonstrate a preference for the CS mechanism; however, the IF mechanism describes the binding of certain ligands to P450 3A4 and P450 101A1 (P450cam) . Nonetheless, even in cases where IF is dominant, the presence of multiple enzyme states prior to ligand binding is not ruled out.…”