CYP2C9 Ile359 and Leu359 variants: enzyme kinetic study with seven substrates.
To assess the effects of Ile359 to Leu359 change on CYP2C9-mediated metabolism, we performed site-directed mutagenesis and cDNA expression in yeast for CYP2C9 and examined in detail the kinetics of seven metabolic reactions by wild-type CYP2C9 (Ile359) and its Leu359 variant. For the metabolism of all the substrates studied, the Leu359 variant exhibited smaller Vmax/Km values than did the wild-type. The differences in the Vmax/Km values between the wild-type and the Leu359 variant varied from 3.4-fold to 26.9-fold. The Leu359 variant had higher Km values than did the wild-type for all the reactions studied. Among the seven reactions studied, the greatest difference in the Vmax values between the wild-type and the Leu359 variant was for piroxicam 5'-hydroxylation (408 versus 19 pmol/min/nmol P450), whereas there were no differences in the Vmax values between the wild-type and the Leu359 variant for diclofenac 4'-hydroxylation and tolbutamide methylhydroxylation. These results indicate that the Ile359 to Leu359 change significantly decreases the catalytic activity of all the CYP2C9-mediated metabolisms studied, whereas the extent of the reduction in activity and changes of the kinetic parameters varies between substrates. Moreover, the amino acid substitution decreased the enantiomeric excess in the formation of 5-(4-hydroxyphenyl)-5-phenylhydantoin from phenytoin.
Takanashi
K
K
Laboratory of Biochemical Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Tainaka
H
H
Kobayashi
K
K
Yasumori
T
T
Hosakawa
M
M
Chiba
K
K
D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D065729:Cytochrome P-450 CYP2C9; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D017931:DNA Primers; D006801:Humans; D007532:Isoleucine / Q000235:genetics*; D007700:Kinetics; D007930:Leucine / Q000235:genetics*; D008861:Microsomes / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D010672:Phenytoin / Q000493:pharmacokinetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000235:genetics* / Q000378:metabolism
D003574:Cytochrome c Group; D017931:DNA Primers; D011994:Recombinant Proteins; D007532:Isoleucine; D010672:Phenytoin; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase; D007930:Leucine