ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 333, No. 2, September 15, pp. 447–458, 1996 Article No. 0414 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 Robert L. Haining,* Ann P. Hunter,* Maurice E. Veronese,† William F. Trager,* and Allan E. Rettie* 1 *Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195; and †Department of Clinical Pharmacology, Flinders University of South Australia, Bedford Park, SA 5042, Australia Received May 1, 1996, and in revised form June 14, 1996 this putative active-site mutation. The greatly de- creased catalytic efficiency of the I359L variant sug- The purpose of the present studies was to define the gests that leucine homozygotes would eliminate (S )- role of the I359L allelic variant of CYP2C9 in the me- warfarin, and probably many other CYP2C9 sub- tabolism of the low therapeutic index anticoagulant strates, at much slower rates in vivo than individuals warfarin, by performing in vitro kinetic studies with expressing the wild-type enzyme.  1996 Academic Press, Inc. the two enantiomers of the drug. To obtain sufficient Key Words: cytochrome P450; expression; baculovi- quantities of these variants to perform kinetic studies rus; warfarin; stereoselectivity; CYP2C9. at physiologically relevant substrate concentrations, methodology was established for the high-level expres- sion, purification, and structural characterization of wild-type CYP2C9 and CYP2C9V1 using the baculovi- rus system. Both forms were expressed at levels up to The cytochromes P450 comprise a superfamily of 250 nmol/liter and purified in 50–55% yield to specific heme-containing enzymes which are distributed ubiq- contents of 13 – 14 nmol holoenzyme/mg protein. The uitously in the animal and plant kingdoms. At least 26 purified preparations were characterized by Edman CYP 2 gene clusters have been mapped to the human degradation and electrospray – mass spectrometry. genome (1). In addition to their endogenous substrates Both forms of the enzyme metabolized the pharmaco- which include steroid hormones, prostaglandins, and logically more potent (S )-enantiomer of warfarin with vitamin D, these monooxygenases are responsible for the same regioselectivity; however, CYP2C9V1 exhib- catalyzing oxidative steps during the metabolism of ited a fivefold lower V max and a fivefold higher K m com- many xenobiotics (2). The tremendous variation and pared to the wild-type enzyme for this substrate. Nei- flexibility in the metabolic capabilities of these isoforms ther form of the enzyme formed significant quantities are probably the result of adaptive, divergent evolution of the (R)-warfarin phenols. Additional studies per- designed to rid organisms of toxic compounds found formed with prochiral arylalkyl sulfides provided con- routinely in foodstuffs (3). firmation of the low turnover rates catalyzed by Oxidative metabolism of xenobiotics is carried out CYP2C9V1 and demonstrated further that sulfoxide principally by members of the CYP1, CYP2, CYP3, and product stereochemistry did not differ significantly CYP4 families, of which the CYP2C subfamily is the between the two variants. Therefore, decreased cata- most complex in mammalian species (4). The human lytic efficiency rather than a gross alteration in sub- CYP2C subfamily consists of four genes: CYP2C8, strate orientation appears to be the consequence of CYP2C9, CYP2C18, and CYP2C19, all of which map This work supported in part by National Institutes of Health Grant 2 Abbreviations used: CYP, cytochrome P450; DTT, dithiothreitol; GM32165. 1 To whom correspondence should be addressed at Department of m.o.i., multiplicity of infection; PEG, polyethylene glycol; p.i., postin- fection; PMSF, phenylmethylsulfonyl fluoride; DLPC, dilaurylphos- Medicinal Chemistry, Box 357610, University of Washington, Seat- tle, WA 98195. Fax: 206-685-3252. phatidylcholine; FBS, fetal bovine serum. 447 0003-9861/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.