Differential roles of Arg97, Asp293, and Arg108 in enzyme stability and substrate specificity of CYP2C9.
CYP2C9 metabolizes a wide range of drugs, many of which are negatively charged at physiological pH. Therefore, it has been thought that complementarily charged amino acid(s) are critically involved in substrate binding. Previous studies have implicated arginine residues at positions 97, 105, and 108 and aspartate at position 293 in the normal catalytic function of the enzyme. To elucidate the role of these amino acids in the substrate specificity of CYP2C9, a series of mutants were constructed and analyzed for functional activity, thermal stability, and ligand binding. Charge-modifying mutations at positions 97, 105, and 293 decreased catalytic activity toward diclofenac, (S)-warfarin, and pyrene in a substrate-independent manner with Arg105 the least, and Arg97 the most, sensitive amino acids in this regard. Decreases in functional activity paralleled thermal instability of the mutants, suggesting that loss of function reflects more generalized structural changes rather than the absence of a specifically charged amino acid at these three positions. The R108H mutant was inactive toward all three substrates because of unexpected nitrogen ligation to the heme. Conversely, the R108F mutant exhibited substrate-dependent catalytic behavior, with almost complete loss of activity toward (S)-warfarin and diclofenac, but preservation of pyrene metabolism. In addition, the R108F mutation abrogated the Type I difference spectra induced by flurbiprofen and benzbromarone, obligate anions at physiological pH. These data identify critical roles for Arg97 and Asp293 in the structural stability of the enzyme and demonstrate a selective role for Arg108 in the binding and metabolism of negatively charged substrates of CYP2C9.
Dickmann
Leslie J
LJ
Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195, USA.
Locuson
Charles W
CW
Jones
Jeffrey P
JP
Rettie
Allan E
AE
D019943:Amino Acid Substitution; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D065729:Cytochrome P-450 CYP2C9; D004008:Diclofenac / Q000378:metabolism; D004795:Enzyme Stability; D015870:Gene Expression; D006639:Histidine / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D011721:Pyrenes / Q000378:metabolism; D013379:Substrate Specificity; D014859:Warfarin / Q000378:metabolism
D011721:Pyrenes; D004008:Diclofenac; D001224:Aspartic Acid; D006639:Histidine; D014859:Warfarin; D001120:Arginine; C030984:pyrene; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases