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

Robert L. Haining, Jeffrey P. Jones, Kirk R. Henne, Michael B. Fisher, Dennis Koop, William F. Trager, Allan E. Rettie

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Abstract

Previous modeling efforts have suggested that coumarin ligand binding to CYP2C9 is dictated by electrostatic and π-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 potential hydrophobic, aromatic active-site residues which could π-stack with the nonmetabolized C-9 phenyl ring of the warfarin enantiomers. To test this hypothesis, we introduced mutations at key residues located in the putative loop region between the B' and C helices of CYP2C9. The F110L, F110Y, V113L, and F114L mutants, but not the F114Y mutant, expressed readily, and the purified proteins were each active in the metabolism of lauric acid. The V113L mutant metabolized: neither (R)- nor (S)-warfarin, and the F114L mutant alone displayed altered metabolite profiles for the warfarin enantiomers. Therefore, the effect of the F110L and F114L mutants on the interaction of CYP2C9 with several of its substrates as well as the potent inhibitor sulfaphenazole was chosen for examination in further detail. For each substrate examined, 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 significant metabolism occurred for the 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 decreased 4-fold and the K(m) was increased 13-fold by the F114L mutation, whereas kinetic parameters for lauric acid metabolism, a substrate which cannot interact with the enzyme by a π-stacking mechanism, were not markedly affected by this mutation. Finally, the F114L mutant effected a greater than 100-fold increase in the K(i) for inhibition of CYP2C9 activity by sulfaphenazole. These data support a role for B'-C helix loop residues F114 and V113 in the hydrophobic binding of warfarin to CYP2C9, and are consistent with π-stacking to F114 for certain aromatic ligands.

Original languageEnglish (US)
Pages (from-to)3285-3292
Number of pages8
JournalBiochemistry
Volume38
Issue number11
DOIs
StatePublished - Mar 16 1999

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Warfarin
Substrate Specificity
Anchors
Binding Sites
lauric acid
Substrates
Enantiomers
Sulfaphenazole
Metabolism
Metabolites
Kinetic parameters
Mutation
Ligands
Hydroxylation
Enzymes
Cytochrome P-450 CYP2C9
Static Electricity
Electrostatics
Catalytic Domain
Proteins

ASJC Scopus subject areas

  • Biochemistry

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Enzymatic determinants of the substrate specificity of CYP2C9 : Role of B'-C loop residues in providing the π-stacking anchor site for warfarin binding. / Haining, Robert L.; Jones, Jeffrey P.; Henne, Kirk R.; Fisher, Michael B.; Koop, Dennis; Trager, William F.; Rettie, Allan E.

In: Biochemistry, Vol. 38, No. 11, 16.03.1999, p. 3285-3292.

Research output: Contribution to journalArticle

Haining, Robert L. ; Jones, Jeffrey P. ; Henne, Kirk R. ; Fisher, Michael B. ; Koop, Dennis ; Trager, William F. ; Rettie, Allan E. / Enzymatic determinants of the substrate specificity of CYP2C9 : Role of B'-C loop residues in providing the π-stacking anchor site for warfarin binding. In: Biochemistry. 1999 ; Vol. 38, No. 11. pp. 3285-3292.
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abstract = "Previous modeling efforts have suggested that coumarin ligand binding to CYP2C9 is dictated by electrostatic and π-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 potential hydrophobic, aromatic active-site residues which could π-stack with the nonmetabolized C-9 phenyl ring of the warfarin enantiomers. To test this hypothesis, we introduced mutations at key residues located in the putative loop region between the B' and C helices of CYP2C9. The F110L, F110Y, V113L, and F114L mutants, but not the F114Y mutant, expressed readily, and the purified proteins were each active in the metabolism of lauric acid. The V113L mutant metabolized: neither (R)- nor (S)-warfarin, and the F114L mutant alone displayed altered metabolite profiles for the warfarin enantiomers. Therefore, the effect of the F110L and F114L mutants on the interaction of CYP2C9 with several of its substrates as well as the potent inhibitor sulfaphenazole was chosen for examination in further detail. For each substrate examined, 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 significant metabolism occurred for the 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 decreased 4-fold and the K(m) was increased 13-fold by the F114L mutation, whereas kinetic parameters for lauric acid metabolism, a substrate which cannot interact with the enzyme by a π-stacking mechanism, were not markedly affected by this mutation. Finally, the F114L mutant effected a greater than 100-fold increase in the K(i) for inhibition of CYP2C9 activity by sulfaphenazole. These data support a role for B'-C helix loop residues F114 and V113 in the hydrophobic binding of warfarin to CYP2C9, and are consistent with π-stacking to F114 for certain aromatic ligands.",
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AU - Henne, Kirk R.

AU - Fisher, Michael B.

AU - Koop, Dennis

AU - Trager, William F.

AU - Rettie, Allan E.

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AB - Previous modeling efforts have suggested that coumarin ligand binding to CYP2C9 is dictated by electrostatic and π-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 potential hydrophobic, aromatic active-site residues which could π-stack with the nonmetabolized C-9 phenyl ring of the warfarin enantiomers. To test this hypothesis, we introduced mutations at key residues located in the putative loop region between the B' and C helices of CYP2C9. The F110L, F110Y, V113L, and F114L mutants, but not the F114Y mutant, expressed readily, and the purified proteins were each active in the metabolism of lauric acid. The V113L mutant metabolized: neither (R)- nor (S)-warfarin, and the F114L mutant alone displayed altered metabolite profiles for the warfarin enantiomers. Therefore, the effect of the F110L and F114L mutants on the interaction of CYP2C9 with several of its substrates as well as the potent inhibitor sulfaphenazole was chosen for examination in further detail. For each substrate examined, 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 significant metabolism occurred for the 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 decreased 4-fold and the K(m) was increased 13-fold by the F114L mutation, whereas kinetic parameters for lauric acid metabolism, a substrate which cannot interact with the enzyme by a π-stacking mechanism, were not markedly affected by this mutation. Finally, the F114L mutant effected a greater than 100-fold increase in the K(i) for inhibition of CYP2C9 activity by sulfaphenazole. These data support a role for B'-C helix loop residues F114 and V113 in the hydrophobic binding of warfarin to CYP2C9, and are consistent with π-stacking to F114 for certain aromatic ligands.

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