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

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 in dex anticoagulant warfarin, by performing in vitro kinetic studies wit h 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-lev el expression, purification, and structural characterization of wild-t ype 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 pharmacol ogically more potent (S)-enantiomer of warfarin with the same regiosel ectivity; however, CYP2C9V1 exhibited a fivefold lower V-max and a fiv efold higher K-m compared to the wild-type enzyme for this substrate, Neither form of the enzyme formed significant quantities of the (R)-wa rfarin phenols. Additional studies performed with prochiral arylalkyl sulfides provided confirmation of the low turnover rates catalyzed by CYP2C9V1 and demonstrated further that sulfoxide product stereochemist ry did not differ significantly between the two variants. Therefore, d ecreased catalytic efficiency rather than a gross alteration in substr ate 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)-warfari n, and probably many other CYP2C9 substrates, at much slower rates in vivo than individuals expressing the wild-type enzyme. (C) 1996 Academ ic Press, Inc.