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J. Biol. Chem., Vol. 281, Issue 14, 9127-9136, April 7, 2006

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Kinetics and Thermodynamics of Ligand Binding by Cytochrome P450 3A4^*

From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

Cytochrome P450 (P450) 3A4, the major catalyst involved in human drug oxidation, displays substrate- and reaction-dependent homotropic and heterotropic 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 -naphthoflavone allowed analysis of an initial ligand-P450 3A4 interaction that does not cause a perturbation of the heme spectrum. 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 dissociates subsequently. A three-step substrate binding model is proposed, based on absorbance and fluorescence stopped-flow kinetic data and equilibrium binding data obtained with bromocriptine, and evaluated using kinetic modeling. The results are consistent with the substrate molecule binding at a site peripheral to the active site and subsequently moving toward the active site to bind to the heme and resulting in a low to high spin iron shift. The last step is attributed to a conformational change in the enzyme active site. The later steps of binding were shown to have rate constants comparable with 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.

Received for publication, October 19, 2005 , and in revised form, January 3, 2006.

^* This work was supported in part by United States Public Health Service Grants R01 CA090426 and P30 ES000267. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a plot of binding of flavone to P450 3A4, second-order plots for binding of the ligands presented in Table 1 to P450 3A4, midazolam binding kinetic data fit to the models shown in Scheme 1, testosterone binding kinetic data fit to a three-step model with two ligands, and sample DynfaFit scripts for bromocriptine binding kinetic data fit to a three-step model.

¹ To whom correspondence should be addressed: Dept. of Biochemistry, Vanderbilt University School of Medicine, 638 Robinson Research Bldg., 23rd and Pierce Aves., Nashville, TN 37232-0146. Tel.: 615-322-2261; Fax: 615-322-3141; E-mail: f.guengerich{at}vanderbilt.edu.

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