Allosteric Modulation of Substrate Motion in Cytochrome P450 3A4-Mediated Xylene Oxidation

Abstract

Many cytochrome P450 enzymes (CYPs) exhibit allosteric behavior reflecting a complex ligand-binding process involving numerous factors: conformational selection, protein-protein interactions, substrate/effector/protein structure, and multiple-ligand binding. The interplay of CYP plasticity and rigidity contributes to substrate/product selectivity and to allosterism. Detailed evidence describing how protein motion modulates product selectivity is incomplete as are descriptions of effector-induced modulation of substrate dynamics. Our intent was to discover details of allosteric behavior and CYP3A4 flexibility and rigidity by investigating substrate motion using low-molecular weight ligands. Steady state kinetics and product ratios were measured for oxidation of m-xylene-(2)H3 and p-xylene; intramolecular isotope effects were measured for m-xylene-(2)H3 oxidation as a function of m-xylene-(2)H3 and p-xylene concentration. Biphasic kinetic plots indicated homotropic cooperative behavior with xylene isomers. Selectivity for aromatic hydroxylation over benzylic hydroxylation of m-xylene-(2)H3 supports a model in which the region near the CYP3A4 active oxidizing species limits substrate dynamics. p-Xylene impedes the motion of m-xylene-(2)H3 substrates that have access to the active oxidizing species: (kH/kD)obs values for m-xylene-(2)H3 decreased with p-xylene concentration. m-Xylene-(2)H3 and p-xylene do not have simultaneous access to the active oxidizing species: deuterium-labeled and unlabeled p-xylene exhibited similar effects on the (kH/kD)obs values for m-xylene-(2)H3 oxidation. p-Xylene and m-xylene-(2)H3 bind at different sites: m-xylene-(2)H3 oxidation rates and product selectivity were consistent across the p-xylene concentration range. Overall, this study indicates that the intramolecular isotope effect experimental design provides a unique opportunity to investigate allosteric mechanisms as it provides information about substrate motion when the enzyme is primed to oxidize substrates.