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Effector-Mediated Alteration of Substrate Orientation in Cytochrome P450 2C9

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Department of Basic Pharmaceutical Sciences, West Virginia University School of Pharmacy, Morgantown, West Virginia 26506, and Department of Natural Sciences and Mathematics, West Liberty State College, West Liberty, West Virginia 26074
Cite this: Biochemistry 2004, 43, 22, 7207–7214
Publication Date (Web):May 8, 2004
https://doi.org/10.1021/bi036158o
Copyright © 2004 American Chemical Society

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    Abstract

    Cytochrome P450 2C9 (CYP2C9)-mediated flurbiprofen 4‘-hydroxylation is activated by the presence of dapsone resulting in reduction of the Km for flurbiprofen hydroxylation and an increase in Vm. Previous spectral binding studies have demonstrated that the binding of flurbiprofen with CYP2C9 is increased (decrease in KS) by the presence of dapsone. We hypothesized that the two compounds are simultaneously in the active site with the presence of dapsone causing flurbiprofen to be oriented more closely to the heme. T1 relaxation rates determined by NMR were used to estimate the distances of protons on these compounds from the paramagnetic heme-iron center. Samples contained 0.014 μM CYP2C9 and 145 μM flurbiprofen in the presence and absence of 100 μM dapsone. Estimated distances of various flurbiprofen protons from the heme ranged from 4.2 to 4.5 Å in the absence of dapsone and from 3.2 to 3.8 Å in the presence of dapsone. The 4‘ proton of flurbiprofen, the site of metabolism, showed one of the greatest differences in distance from the heme in the presence of dapsone, 3.50 Å, as compared to the absence of dapsone, 4.41 Å. Dapsone protons were less affected, being 4.40 Å from the heme in the absence of flurbiprofen and 4.00−4.01 Å from the heme in the presence of flurbiprofen. Molecular modeling studies were also performed to corroborate the relative orientations of flurbiprofen and dapsone in the active site of CYP2C9. Shift of the 4‘ proton of flurbiprofen closer to the heme iron of CYP2C9 in the presence of dapsone may play a role in activation.

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     This work was supported in part by grants from the Public Health Service (Grant GM-63215 to T.S.T), National Center for Research Resources (Grant 1P20RR16477 to J.S.A.) and NSF (Grant 1002165R to P.M.G.).

     West Virginia University School of Pharmacy.

     Current Address:  Department of Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455.

    §

     West Liberty State College.

    *

     Corresponding Author. Mailing address:  Department of Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota, 7-168 Weaver-Densford Hall, 308 Harvard St. SE, Minneapolis, MN 55455. Tel:  612-625-7665. Fax:  612-625-3927. E-mail:  [email protected].

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