Kinetics and thermodynamics of ligand binding by cytochrome P450 3A4 and relevance to substrate cooperativity

Cytochrome P450 (P450) 3A4, the major catalyst involved in human drug oxidation, displays substrate and reaction dependent cooperative behavior. Although several models have been proposed, these mainly rely on steady‐state kinetics and do not provide information on the contribution of the individual steps of P450 catalytic cycle to the observed cooperativity. In this work, we focused on the kinetics of substrate binding, and the fluorescent properties of bromocriptine and α‐naphthoflavone allowed analysis of an initial ligand‐P450 3A4 interaction that does not cause a perturbation of the heme spectrum. A three‐step substrate binding model is proposed, based on absorbance and fluorescence stopped‐flow kinetic data, and evaluated using kinetic modeling. The results are consistent with the first substrate molecule binding at a site peripheral to the active site, followed by a second ligand molecule binding to the heme. The last step is attributed to a conformational change in the enzyme active site. The binding stoichiometry for bromocriptine was determined to be unity using isothermal titration calorimetry and equilibrium dialysis methods, suggesting that the ligand bound to the peripheral site during the initial encounter may subsequently dissociate. The later steps of binding were shown to have rate constants comparable to the subsequent steps of the catalytic cycle. The P450 3A4 binding process is more complex than a two‐state system and the overlap of rates of some of the events with subsequent steps is proposed to underlie the observed cooperativity. Supported in part by USPHS R01 CA90426 and P30 ES00267