title: Assessment of arginine 97 and lysine 72 as determinants of substrate specificity in cytochrome P450 2C9 (CYP2C9).
abstract: CYP2C9 is distinguished by a preference for substrates bearing a negative charge at physiological pH. Previous studies have suggested that CYP2C9 residues R97 and K72 may play roles in determining preference for anionic substrates by interaction at the active site or in the access channel. The aim of the present study was to assess the role of these two residues in determining substrate selectivity. R97 and K72 were substituted with negative, uncharged polar and hydrophobic residues using a degenerate polymerase chain reaction-directed strategy. Wild-type and mutant enzymes were expressed in bicistronic format with human cytochrome P450 reductase in Escherichia coli. Mutation of R97 led to a loss of holoenzyme expression for R97A, R97V, R97L, R97T, and R97E mutants. Low levels of hemoprotein were detected for R97Q, R97K, R97I, and R97P mutants. Significant apoenzyme was observed, suggesting that heme insertion or protein stability was compromised in R97 mutants. These observations are consistent with a structural role for R97 in addition to any role in substrate binding. By contrast, all K72 mutants examined (K72E, K72Q, K72V, and K72L) could be expressed as hemoprotein at levels comparable to wild-type. Type I binding spectra were obtained with wild-type and K72 mutants using diclofenac and ibuprofen. Mutation of K72 had little or no effect on the interaction with these substrates, arguing against a critical role in determining substrate specificity. Thus, neither residue appears to play a role in determining substrate specificity, but a structural role for R97 can be proposed consistent with recently published crystallographic data for CYP2C9 and CYP2C5.
authors.affiliation: Department of Physiology and Pharmacology, School of Biomedical Sciences, The University of Queensland, St. Lucia, Australia, 4072.
authors.forename: Carwyn
authors.initials: C
authors.lastname: Davies
authors.forename: Katey
authors.initials: K
authors.lastname: Witham
authors.forename: Justin R
authors.initials: JR
authors.lastname: Scott
authors.forename: Andrew
authors.initials: A
authors.lastname: Pearson
authors.forename: James J
authors.initials: JJ
authors.lastname: DeVoss
authors.forename: Sandra E
authors.initials: SE
authors.lastname: Graham
authors.forename: Elizabeth M J
authors.initials: EM
authors.lastname: Gillam
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D065729:Cytochrome P-450 CYP2C9; D004008:Diclofenac / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D006420:Hemeproteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D020035:Holoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007052:Ibuprofen / Q000378:metabolism; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000187:drug effects / Q000502:physiology; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D009288:Naproxen / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity / Q000187:drug effects / Q000502:physiology*
chemical_list: D006420:Hemeproteins; D020035:Holoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D004008:Diclofenac; D009288:Naproxen; D001120:Arginine; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D009251:NADPH-Ferrihemoprotein Reductase; D008239:Lysine; D007052:Ibuprofen