Enzymatic determinants of the substrate specificity of CYP2C9: Role of B '-C loop residues in providing the pi-stacking anchor site for warfarin binding

Citation
Rl. Haining et al., Enzymatic determinants of the substrate specificity of CYP2C9: Role of B '-C loop residues in providing the pi-stacking anchor site for warfarin binding, BIOCHEM, 38(11), 1999, pp. 3285-3292
Citations number
29
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
00062960 → ACNP
Volume
38
Issue
11
Year of publication
1999
Pages
3285 - 3292
Database
ISI
SICI code
0006-2960(19990316)38:11<3285:EDOTSS>2.0.ZU;2-R
Abstract
Previous modeling efforts have suggested that coumarin ligand binding to CY P2C9 is dictated by electrostatic and pi-stacking interactions with complem entary amino acids of the protein. In this study, analysis of a combined Co MFA-homology model for the enzyme identified F110 and F114 as potential hyd rophobic, aromatic active-site residues which could pi-stack with the nonme tabolized C-9 phenyl ring of the warfarin enantiomers. To test this hypothe sis, we introduced mutations at key residues located in the putative loop r egion between the B' and C helices of CYP2C9. The F110L, F110Y, V113L, and F114L mutants, but not the F114Y mutant, expressed readily, and the purifie d proteins were each active in the metabolism of lauric acid. The V113L mut ant metabolized neither (R)- nor (S)-warfarin, and the F114L mutant alone d isplayed altered metabolite profiles for the warfarin enantiomers. Therefor e, the effect of the F110L and F114L mutants on the interaction of CYP2C9 w ith several of its substrates as well as the potent inhibitor sulfaphenazol e was chosen for examination in further detail. For each substrate examined , the F110L mutant exhibited modest changes in its kinetic parameters and p roduct profiles. However, the F114L mutant altered the metabolite ratios fo r the warfarin enantiomers such that significant metabolism occurred for th e first time on the putative C-9 phenyl anchor, at the 4'-position of (R)- and (S)-warfarin. In addition, the V-max for (S)-warfarin 7-hydroxylation d ecreased 4-fold and the K-m was increased 13-fold by the F114L mutation, wh ereas kinetic parameters for lauric acid metabolism, a substrate which cann ot interact with the enzyme by a pi-stacking mechanism, were not markedly a ffected by this mutation. Finally, the F114L mutant effected a greater than 100-fold increase in the K-i for inhibition of CYP2C9 activity by sulfaphe nazole. These data support a role for B'-C helix loop residues F114 and V11 3 in the hydrophobic binding of warfarin to CYP2C9, and are consistent with pi-stacking to F114 for certain aromatic ligands.