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

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 <i>m</i>-xylene-²H₃ and <i>p</i>-xylene; intramolecular isotope effects were measured for <i>m</i>-xylene-²H₃ oxidation as a function of <i>m</i>-xylene-²H₃ and <i>p</i>-xylene concentration. Biphasic kinetic plots indicated homotropic cooperative behavior with xylene isomers. Selectivity for aromatic hydroxylation over benzylic hydroxylation of <i>m</i>-xylene-²H₃ supports a model in which the region near the CYP3A4 active oxidizing species limits substrate dynamics. <i>p</i>-Xylene impedes the motion of <i>m</i>-xylene-²H₃ substrates that have access to the active oxidizing species: (<i>k</i>_H/<i>k</i>_D)_obs values for <i>m</i>-xylene-²H₃ decreased with <i>p</i>-xylene concentration. <i>m</i>-Xylene-²H₃ and <i>p</i>-xylene do not have simultaneous access to the active oxidizing species: deuterium-labeled and unlabeled <i>p</i>-xylene exhibited similar effects on the (<i>k</i>_H/<i>k</i>_D)_obs values for <i>m</i>-xylene-²H₃ oxidation. <i>p</i>-Xylene and <i>m</i>-xylene-²H₃ bind at different sites: <i>m</i>-xylene-²H₃ oxidation rates and product selectivity were consistent across the <i>p</i>-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.