CYP2C9 Ile359 and Leu359 variants:: enzyme kinetic study with seven substrates

To assess the effects of Ile(359) to Leu(359) 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 Leu(359) variant. For the metabolism of all the substrates studied, the Leu(359) Variant exhibited smaller V-max/K-m values than did the wild-type. The differences in the Vmax/Km values between the wild-type and the Leu(359) variant varied from 3.4-fold to 26.9-fold. The Leu(359) variant had higher K-m values than did the wild-type for all the reactions studied. Among the seven reactions studied, the greatest difference in the V-max values between the wild-type and the Leu(359) variant was for piroxicam 5'-hydroxylation (408 versus 19 pmol/ min/nmol P450), whereas there were no differences in the V-max values between the wildtype and the Leu(359) Variant for diclofenac 4'-hydroxylation and tolbutamide methylhydroxylation, These results indicate that the Ile(359) to Leu(359) 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, Pharmacogenetics 10:95-104 (C) 2000 Lippincott Williams & Wilkins.