Abstract
Phenytoin has been an effective anticonvulsant agent for over 60 years, although its clinical use is complicated by nonlinear pharmacokinetics, a narrow therapeutic index, and metabolically based drug-drug interactions. Although it is well established that CYP2C9 is the major cytochrome P450 enzyme controlling metabolic elimination of phenytoin through its oxidative conversion to (S)-5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH), nothing is known about the amino acid binding determinants within the CYP2C9 active site that promote metabolism and maintain the tight stereocontrol of hydroxy metabolite formation. This knowledge gap was addressed here through the construction of nine active site mutants at amino acid positions Phe100, Arg108, Phe114, Leu208, and Phe476 and in vitro analysis of the steady-state kinetics and stereochemistry of p-HPPH formation. The F100L and F114W mutants exhibited 4- to 5-fold increases in catalytic efficiency, whereas the F100W, F114L, F476L, and F476W mutants lost >90% of their phenytoin hydroxylation capacity. This pattern of effects differs substantially from that found previously for (S)-warfarin and (S)-flurbiprofen metabolism, suggesting that these three ligands bind within discrete locations in the CYP2C9 active site. Only the F114L, F476L, and L208V mutants altered phenytoin's orientation during catalytic turnover. The L208V mutant also uniquely demonstrated enhanced 6-hydroxylation of (S)-warfarin. These latter data provide the first experimental evidence for a role of the F-G loop region in dictating the catalytic orientation of substrates within the CYP2C9 active site.
Footnotes
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This work was supported by the National Institutes of Health National Institute of General Medical Sciences [Grants GM32165, GM07750].
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Part of this work was previously presented as follows: Mosher CM and Rettie AE (2006) CYP2C9 active site determinants of phenytoin metabolism, orientation, and lansoprazole-mediated activation. 14th North American Meeting of the International Society for the Study of Xenobiotics; 2006 Oct 22–26; San Juan, Puerto Rico. International Society for the Study of Xenobiotics, Washington, DC; and Tai G (2006) Structural Determinants of CYP2C9's Genetic Variability, Substrate Specificity and Dioxygen Cleavage. Ph.D. Thesis, University of Washington, Seattle, WA.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.109.150706.
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ABBREVIATIONS: P450, cytochrome P450; p-HPPH, 5-(4-hydroxyphenyl)-5-phenylhydantoin; SRS, substrate recognition site; DLPC, dilaurylphosphatidylcholine; LC, liquid chromatography; MS, mass spectrometry; MRM, multiple reaction monitoring; HPPH, 5-hydroxyphenyl-5-phenylhydantoin; pdb, Protein Data Bank.
- Received January 19, 2009.
- Accepted March 2, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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