Allelic Variants of Human Cytochrome P450 2C9: Baculovirus-Mediated Expression, Purification, Structural Characterization, Substrate Stereoselectivity, and Prochiral Selectivity of the Wild-Type and I359L Mutant Forms

• Published in: Archives of biochemistry and biophysics Vol. 333; no. 2; pp. 447 - 458
• Main Authors: Haining, Robert L., Hunter, Ann P., Veronese, Maurice E., Trager, William F., Rettie, Allan E.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.09.1996
• Subjects: Alleles; Amino Acid Sequence; Animals; Aryl Hydrocarbon Hydroxylases; Baculoviridae; baculovirus; Chromatography, Ion Exchange; CYP2C9; Cytochrome P-450 CYP2C9; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - isolation & purification; Cytochrome P-450 Enzyme System - metabolism; cytochrome P450; expression; Genetic Variation; Humans; Insecta; Kinetics; Mass Spectrometry; Molecular Sequence Data; Point Mutation; Rabbits; Rats; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Stereoisomerism; stereoselectivity; Steroid 16-alpha-Hydroxylase; Steroid Hydroxylases - chemistry; Steroid Hydroxylases - isolation & purification; Steroid Hydroxylases - metabolism; Substrate Specificity; Transfection; warfarin; Warfarin - metabolism; CYP2C9; baculovirus; expression; cytochrome P450; stereoselectivity; warfarin
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1996.0414

The purpose of the present studies was to define the role of the I359L allelic variant of CYP2C9 in the metabolism of the low therapeutic index anticoagulant warfarin, by performingin vitrokinetic studies with the two enantiomers of the drug. To obtain sufficient quantities of these variants to perform kinetic studies at physiologically relevant substrate concentrations, methodology was established for the high-level expression, purification, and structural characterization of wild-type CYP2C9 and CYP2C9V1 using the baculovirus system. Both forms were expressed at levels up to 250 nmol/liter and purified in 50–55% yield to specific contents of 13–14 nmol holoenzyme/mg protein. The purified preparations were characterized by Edman degradation and electrospray–mass spectrometry. Both forms of the enzyme metabolized the pharmacologically more potent (S)-enantiomer of warfarin with the same regioselectivity; however, CYP2C9V1 exhibited a fivefold lowerVmaxand a fivefold higherKmcompared to the wild-type enzyme for this substrate. Neither form of the enzyme formed significant quantities of the (R)-warfarin phenols. Additional studies performed with prochiral arylalkyl sulfides provided confirmation of the low turnover rates catalyzed by CYP2C9V1 and demonstrated further that sulfoxide product stereochemistry did not differ significantly between the two variants. Therefore, decreased catalytic efficiency rather than a gross alteration in substrate orientation appears to be the consequence of this putative active-site mutation. The greatly decreased catalytic efficiency of the I359L variant suggests that leucine homozygotes would eliminate (S)-warfarin, and probably many other CYP2C9 substrates, at much slower ratesin vivothan individuals expressing the wild-type enzyme.