Enzymatic Determinants of the Substrate Specificity of CYP2C9: Role of B'-C Loop Residues in Providing the -Stacking Anchor Site for Wa
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- 作者：Robert L. Haining ; Jeffrey P. Jones ; Kirk R. Henne ; Michael B. Fisher ; Dennis R. Koop ; William F. Trager ; and Allan E. Rettie
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：March 16, 1999
- 年：1999
- 卷：38
- 期：11
- 页码：3285 - 3292
- 全文大小：164K
- 年卷期：v.38,no.11(March 16, 1999)
- ISSN：1520-4995
文摘
Previous modeling efforts have suggested that coumarin ligand binding to CYP2C9 is dictatedby electrostatic and 
-stacking interactions with complementary amino acids of the protein. In this study,analysis of a combined CoMFA-homology model for the enzyme identified F110 and F114 as potentialhydrophobic, aromatic active-site residues which could -stack with the nonmetabolized C-9 phenyl ringof the warfarin enantiomers. To test this hypothesis, we introduced mutations at key residues located inthe putative loop region between the B' and C helices of CYP2C9. The F110L, F110Y, V113L, andF114L mutants, but not the F114Y mutant, expressed readily, and the purified proteins were each activein the metabolism of lauric acid. The V113L mutant metabolized neither (R)- nor (S)-warfarin, and theF114L mutant alone displayed altered metabolite profiles for the warfarin enantiomers. Therefore, theeffect of the F110L and F114L mutants on the interaction of CYP2C9 with several of its substrates aswell as the potent inhibitor sulfaphenazole was chosen for examination in further detail. For each substrateexamined, the F110L mutant exhibited modest changes in its kinetic parameters and product profiles.However, the F114L mutant altered the metabolite ratios for the warfarin enantiomers such that significantmetabolism occurred for the first time on the putative C-9 phenyl anchor, at the 4'-position of (R)- and(S)-warfarin. In addition, the Vmax for (S)-warfarin 7-hydroxylation decreased 4-fold and the Km wasincreased 13-fold by the F114L mutation, whereas kinetic parameters for lauric acid metabolism, a substratewhich cannot interact with the enzyme by a -stacking mechanism, were not markedly affected by thismutation. Finally, the F114L mutant effected a greater than 100-fold increase in the Ki for inhibition ofCYP2C9 activity by sulfaphenazole. These data support a role for B'-C helix loop residues F114 andV113 in the hydrophobic binding of warfarin to CYP2C9, and are consistent with -stacking to F114 forcertain aromatic ligands.
-stacking interactions with complementary amino acids of the protein. In this study,analysis of a combined CoMFA-homology model for the enzyme identified F110 and F114 as potentialhydrophobic, aromatic active-site residues which could -stack with the nonmetabolized C-9 phenyl ringof the warfarin enantiomers. To test this hypothesis, we introduced mutations at key residues located inthe putative loop region between the B' and C helices of CYP2C9. The F110L, F110Y, V113L, andF114L mutants, but not the F114Y mutant, expressed readily, and the purified proteins were each activein the metabolism of lauric acid. The V113L mutant metabolized neither (R)- nor (S)-warfarin, and theF114L mutant alone displayed altered metabolite profiles for the warfarin enantiomers. Therefore, theeffect of the F110L and F114L mutants on the interaction of CYP2C9 with several of its substrates aswell as the potent inhibitor sulfaphenazole was chosen for examination in further detail. For each substrateexamined, the F110L mutant exhibited modest changes in its kinetic parameters and product profiles.However, the F114L mutant altered the metabolite ratios for the warfarin enantiomers such that significantmetabolism occurred for the first time on the putative C-9 phenyl anchor, at the 4'-position of (R)- and(S)-warfarin. In addition, the Vmax for (S)-warfarin 7-hydroxylation decreased 4-fold and the Km wasincreased 13-fold by the F114L mutation, whereas kinetic parameters for lauric acid metabolism, a substratewhich cannot interact with the enzyme by a -stacking mechanism, were not markedly affected by thismutation. Finally, the F114L mutant effected a greater than 100-fold increase in the Ki for inhibition ofCYP2C9 activity by sulfaphenazole. These data support a role for B'-C helix loop residues F114 andV113 in the hydrophobic binding of warfarin to CYP2C9, and are consistent with -stacking to F114 forcertain aromatic ligands.