Abstract

Three-dimensional homology models of cytochromes P450 (P450) 2B1 and P450 3A4 have been utilized along with site-directed mutagenesis to elucidate the molecular determinants of substrate specificity. Most of the key residues identified in 2B enzymes fall within five substrate recognition sites (SRSs) and have counterparts in bacterial P450 residues that regulate substrate binding or access. Docking of inhibitors into 2B models has provided a plausible explanation for changes in susceptibility to mechanism-based inactivation that accompany particular amino acid side-chain replacements. These studies provide a basis for predicting drug interactions due to P450 inhibition and for rational inhibitor design. In addition, the location of P450 3A4 residues capable of influencing homotropic stimulation by substrates and heterotropic stimulation by flavonoids has been identified. Steroid hydroxylation by the wild-type enzyme exhibits sigmoidal kinetics, indicative of positive cooperativity. Based on the 3A4 model and single-site mutants, a double mutant in SRS-2 has been constructed that exhibits normal Michaelis-Menten kinetics. Results of modeling and mutagenesis studies suggest that the substrate and effector bind at adjacent sites within a single large cavity in P450 3A4. A thorough understanding of the location and structural requirements of the substrate-binding and effector sites in cytochrome P450 3A4 should prove valuable in rationalizing and predicting interactions among the multitude of drugs and other compounds that bind to the enzyme.