Arginines 97 and 108 in CYP2C9 Are Important Determinants of the Catalytic Function

doi:10.1006/bbrc.2000.2538

Biochemical and Biophysical Research Communications

Volume 270, Issue 3, 21 April 2000, Pages 983-987

https://doi.org/10.1006/bbrc.2000.2538 Get rights and content

Abstract

Human cytochrome P450 2C9 (CYP2C9) is one of the major drug metabolising enzymes which exhibits a broad substrate specificity. The B-C loop is located in the active-site but has been difficult to model, owing to its diverse and flexible structure. To elucidate the function of the B-C loop we used homology modelling based on the Cyp102 structure in combination with functional studies of mutants using diclofenac as a model substrate for CYP2C9. The study shows the importance of the conserved arginine in position 97 and the arginine in position 108 for the catalytic function. The R97A mutant had a 13-fold higher K_m value while the V_max was in the same order as the wild type. The R108 mutant had a 100-fold lower activity with diclofenac compared to the wild-type enzyme. The other six mutants (S95A, F100A, L102A, E104A, R105A, and N107A) had kinetic parameters similar to the CYP2C9 wild-type. Our homology model based on the CYP102 structure as template indicates that R97, L102, and R105 are directed into the active site, whereas R108 is not. The change in catalytic function when arginine 97 was replaced with alanine and the orientation of this amino acid in our homology model indicates its importance for substrate interaction.

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Identification of cytochrome P450 2C18 and 2C76 in tree shrews: P450 2C18 effectively oxidizes typical human P450 2C9/2C19 chiral substrates warfarin and omeprazole with less stereoselectivity
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Cytochromes P450 (P450s or CYPs), especially the CYP2C family, are important drug-metabolizing enzymes that play major roles in drug metabolism. Tree shrews, a non-rodent primate-like species, are used in various fields of biomedical research, notably hepatitis virus infection; however, its drug-metabolizing enzymes have not been fully investigated. In this study, tree shrew CYP2C18, CYP2C76a, CYP2C76b, and CYP2C76c cDNAs were identified and contained open reading frames of 489 or 490 amino acids with high sequence identities (70–78 %) to human CYP2Cs. Tree shrew CYP2C76a, CYP2C76b, and CYP2C76c showed higher sequence identities (79–80 %) to cynomolgus CYP2C76 and were not orthologous to any human CYP2C. Phylogenetic analysis revealed that tree shrew CYP2C18 and CYP2C76s were closely related to rat CYP2Cs and cynomolgus CYP2C76, respectively. Tree shrew CYP2C genes formed a gene cluster similar to human CYP2C genes. All four tree shrew CYP2C mRNAs showed predominant expressions in liver, among the tissue types examined; expression of CYP2C18 mRNA was also detected in small intestine. In liver, CYP2C18 mRNA was the most abundant among the tree shrew CYP2C mRNAs. In metabolic assays using human CYP2C substrates, all tree shrew CYP2Cs showed metabolic activities toward diclofenac, R,S-omeprazole, paclitaxel, and R,S-warfarin, with the activity of CYP2C18 exceeding that of the other CYP2Cs. Moreover, tree shrew CYP2C76 enzymes metabolized progesterone more efficiently than human, cynomolgus, or marmoset CYP2Cs. Therefore, these novel tree shrew CYP2Cs are expressed abundantly in liver, encode functional enzymes that metabolize human CYP2C substrates, and are likely responsible for drug clearances.
Structural characterization of human cytochrome P450 2C19: Active site differences between P450s 2C8, 2C9, and 2C19
2012, Journal of Biological Chemistry
Citation Excerpt :
The presence of Arg-108 in the active site of P450 2C9 is likely to contribute to the high binding affinity observed for benzbromarone and other ionized phenols (40). Mutagenesis studies indicate that substitution of alanine for Arg-108 of P450 2C9 dramatically reduces diclofenac 4-hydroxylase activity (41). Similarly, substitution of a phenylalanine for Arg-108 leads to an almost complete loss of diclofenac and S-warfarin hydroxylation by P450 2C9 while retaining its capacity to catalyze hydroxylation of the nonpolar substrate, pyrene (42).
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Citation Excerpt :
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^☆: Abbreviation used: CYP, cytochrome P450.

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Regular ArticleArginines 97 and 108 in CYP2C9 Are Important Determinants of the Catalytic Function☆

Abstract

Drug Discov. Today

Drug Discov. Today

J. Mol. Biol.

Mol. Cell

Structure

J. Biol. Chem.

Arch. Biochem. Biophys.

Anal. Biochem.

J. Biol. Chem.

J. Biol. Chem.

Drug Metab. Rev.

Br. J. Clin. Pharmacol.

Drug Metab. Disp.

Regular Article
Arginines 97 and 108 in CYP2C9 Are Important Determinants of the Catalytic Function☆