title: Identification of amino acid substitutions that confer a high affinity for sulfaphenazole binding and a high catalytic efficiency for warfarin metabolism to P450 2C19.
abstract: Human cytochrome P450s 2C9 and 2C19 metabolize many important drugs including tolbutamide, phenytoin, and (S)-warfarin. Although they differ at only 43 of 490 amino acids, sulfaphenazole (SFZ) is a potent and selective inhibitor of P450 2C9 with an IC50 and a spectrally determined binding constant, KS, of <1 microM. P450 2C19 is not affected by SFZ at concentrations up to 100 microM. A panel of CYP2C9/2C19 chimeric proteins was constructed in order to identify the sequence differences that underlie this difference in SFZ binding. Replacement of amino acids 227-338 in 2C19 with the corresponding region of 2C9 resulted in high-affinity SFZ binding (KS approximately 4 microM) that was not seen when a shorter fragment of 2C9 was substituted (227-282). However, replacement of amino acids 283-338 resulted in extremely low holoenzyme expression levels in Escherichia coli, indicating protein instability. A single mutation, E241K, which homology modeling indicated would restore a favorable charge pair interaction between K241 in helix G and E288 in helix I, led to successful expression of this chimera that exhibited a KS < 10 microM for SFZ. Systematic replacement of the remaining differing amino acids revealed that two amino acid substitutions in 2C19 (N286S, I289N) confer high-affinity SFZ binding (KS < 5 microM). When combined with a third substitution, E241K, the resulting 2C19 triple mutant exhibited a high cataltyic efficiency for warfarin metabolism with the relaxed stereo- and regiospecificity of 2C19 and a lower KM for (S)-warfarin metabolism (<10 microM) typical of 2C9.
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: F
authors.initials: F
authors.lastname: Jung
authors.forename: K J
authors.initials: KJ
authors.lastname: Griffin
authors.forename: W
authors.initials: W
authors.lastname: Song
authors.forename: T H
authors.initials: TH
authors.lastname: Richardson
authors.forename: M
authors.initials: M
authors.lastname: Yang
authors.forename: E F
authors.initials: EF
authors.lastname: Johnson
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D001189:Aryl Hydrocarbon Hydroxylases; D001667:Binding, Competitive / Q000235:genetics; D002384:Catalysis; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D065731:Cytochrome P-450 CYP2C19; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D013237:Stereoisomerism; D013426:Sulfaphenazole / Q000378:metabolism*; D014859:Warfarin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D013426:Sulfaphenazole; D014859:Warfarin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19