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J. Biol. Chem., Vol. 283, Issue 6, 3487-3496, February 8, 2008

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CYP2E1 Substrate Inhibition

MECHANISTIC INTERPRETATION THROUGH AN EFFECTOR SITE FOR MONOCYCLIC COMPOUNDS^*

Samuel L. Collom, Ryan M. Laddusaw, Amber M. Burch, Petr Kuzmic^¶, Martin D. Perry, Jr., and Grover P. Miller¹

From the Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, the Department of Chemistry, Ouachita Baptist University, Arkadelphia, Arkansas 71998, and ^¶BioKin, Ltd., Pullman, Washington 99163

In this study we offer a mechanistic interpretation of the previously known but unexplained substrate inhibition observed for CYP2E1. At low substrate concentrations, p-nitrophenol (pNP) was rapidly turned over (47 min^-1) with relatively low K_m (24 µM); nevertheless, at concentrations of >100 µM, the rate of pNP oxidation gradually decreased as a second molecule bound to CYP2E1 through an effector site (K_ss = 260 µM), which inhibited activity at the catalytic site. 4-Methylpyrazole (4MP) was a potent inhibitor for both sites through a mixed inhibition mechanism. The K_i for the catalytic site was 2.0 µM. Although we were unable to discriminate whether an EIS or ESI complex formed, the respective inhibition constants were far lower than K_ss. Bicyclic indazole (IND) inhibited catalysis through a single CYP2E1 site (K_i = 0.12 µM). Similarly, 4MP and IND yielded type II binding spectra that reflected the association of either two 4MP or one IND molecule(s) to CYP2E1, respectively. Based on computational docking studies with a homology model for CYP2E1, the two sites for monocyclic molecules, pNP and 4MP, exist within a narrow channel connecting the active site to the surface of the enzyme. Because of the presence of the heme iron, one site supports catalysis, whereas the other more distal effector site binds molecules that can influence the binding orientation and egress of molecules for the catalytic site. Although IND did not bind these sites simultaneously, the presence of IND at the catalytic site blocked binding at the effector site.

Received for publication, September 11, 2007 , and in revised form, December 4, 2007.

^* 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 the scripts (input data) for the DynaFit software (15, 16).

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 W. Markham St. Slot 516, Little Rock, AR 72205. Tel.: 501-526-6486; Fax: 501-686-8169; E-mail: millergroverp{at}uams.edu.

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