NDLI: Functional Analysis of Phenylalanine Residues in the Active Site of CYP2C9†

Content Provider	PubMed Central
Author	Mosher, Carrie M. Hummel, Matthew A. Tracy, Timothy S. Rettie, Allan E.
Abstract	The two published crystal structures of CYP2C9, complexed with (S)-warfarin or flurbiprofen, implicate a cluster of three active-site phenylalanine residues (F100, F114, F476) in ligand binding. However, these three residues appear to interact differently with these two ligands based on the static crystal structures. To elucidate the importance of CYP2C9’s active site phenylalanines on substrate binding, orientation and catalytic turnover, a series of leucine and tryptophan mutants was constructed and their interactions with (S)-warfarin and (S)-flurbiprofen examined. The F100→L mutation had minor effects on substrate binding and metabolism of each substrate. In contrast, the F114L and F476L mutants exhibited substantially reduced (S)-warfarin metabolism and altered hydroxy metabolite profiles, but only modestly decreased NSAID turnover, while maintaining product regioselectivity. The F114→W and F476→W mutations also had opposing effects on (S)-warfarin versus NSAID turnover. Notably, the F476W mutant increased the efficiency of (S)-warfarin metabolism 5-fold, yet decreased the efficiency of (S)-flurbiprofen turnover 20-fold. 1H-NMR T 1 relaxation studies suggested a slightly closer positioning of (S)-warfarin to the heme in the F476W mutant relative to the wild-type enzyme, and stoichiometry studies indicated enhanced coupling of reducing equivalents to product formation for (S)-warfarin, again in contrast to effects observed with (S)-flurbiprofen. These data demonstrate that F114 and F476, but not F100, influence (S)-warfarin’s catalytic orientation. Differential interactions of F476 mutants with the two substrates suggest that their catalytically productive binding modes are not superimposable.
Related Links	http://dx.doi.org/10.1021/bi801231m
Ending Page	11734
Page Count	10
Starting Page	11725
File Format	PDF
ISSN	00062960
e-ISSN	15204995
Journal	Biochemistry
Issue Number	45
Volume Number	47
Language	English
Publisher Date	2008-11-11
Access Restriction	Open
Subject Keyword	Biochemistry Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Biochemistry

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