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J. Biol. Chem., Vol. 282, Issue 9, 6863-6874, March 2, 2007

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Multiple Sequential Steps Involved in the Binding of Inhibitors to 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 is an extensively studied human enzyme involved in the metabolism of >50% of drugs. The mechanism of the observed homotropic and heterotropic cooperativity in P450 3A4-catalyzed oxidations is not well understood, and together with the cooperative behavior, a detailed understanding of interaction of drug inhibitors with P450 3A4 is important in predicting clinical drug-drug interactions. The interactions of P450 3A4 with several structurally diverse inhibitors were investigated using both kinetic and thermodynamic approaches to resolve the steps involved in binding of these ligands. The results of pre-steady-state absorbance and fluorescence experiments demonstrate that inhibitor binding is clearly a multistep process, even more complex than the binding of substrates. Based on spectrophotometric equilibrium binding titrations as well as isothermal titration calorimetry experiments, the stoichiometry of binding appears to be 1:1 in the concentration ranges studied. Using a sequential-mixing stopped-flow approach, we were also able to show that the observed multiphasic binding kinetics is the result of sequential events as opposed to the existence of multiple enzyme populations in dynamic equilibrium that interact with ligands at different rates. We propose a three-step minimal model for inhibitor binding, developed with kinetic simulations, consistent with our previously reported model for the binding of substrates, although it is possible that even more steps are involved.

Received for publication, November 6, 2006 , and in revised form, December 13, 2006.

^* This work was supported in part by United States Public Health Service Grants R37 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 three-dimensional structures of inhibitor molecules, itraconazole binding kinetic data fit to a bi-exponential and a tri-exponential equation, ketoconazole binding kinetics data fit to the model shown in Scheme 1E, and sample DynaFit scripts and outputs for ketoconazole binding kinetic data fit to the model shown in Scheme 1A.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Center in Molecular Toxicology, 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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				C. D. Sohl, E. M. Isin, R. L. Eoff, G. A. Marsch, D. F. Stec, and F. P. Guengerich Cooperativity in Oxidation Reactions Catalyzed by Cytochrome P450 1A2: HIGHLY COOPERATIVE PYRENE HYDROXYLATION AND MULTIPHASIC KINETICS OF LIGAND BINDING J. Biol. Chem., March 14, 2008; 283(11): 7293 - 7308. [Abstract] [Full Text] [PDF]