Takanashi K, Tainaka H, Kobayashi K, Yasumori T, Hosakawa M, Chiba K
Laboratory of Biochemical Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Pharmacogenetics. 2000 Mar;10(2):95-104. doi: 10.1097/00008571-200003000-00001.
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.
为评估异亮氨酸359突变为亮氨酸359对CYP2C9介导的代谢作用的影响，我们进行了定点诱变，并在酵母中对CYP2C9进行了cDNA表达，详细研究了野生型CYP2C9（异亮氨酸359）及其亮氨酸359变体对7种代谢反应的动力学。对于所有研究的底物代谢，亮氨酸359变体的Vmax/Km值均小于野生型。野生型和亮氨酸359变体之间Vmax/Km值的差异在3.4倍至26.9倍之间。对于所有研究的反应，亮氨酸359变体的Km值均高于野生型。在所研究的7种反应中，野生型和亮氨酸359变体之间Vmax值差异最大的是吡罗昔康5'-羟基化反应（分别为408与19 pmol/分钟/纳摩尔P450），而对于双氯芬酸4'-羟基化反应和甲苯磺丁脲甲基羟基化反应，野生型和亮氨酸359变体之间的Vmax值没有差异。这些结果表明，异亮氨酸359突变为亮氨酸359显著降低了所有所研究的CYP2C9介导的代谢反应的催化活性，而活性降低的程度和动力学参数的变化在不同底物之间有所不同。此外，氨基酸替换降低了苯妥英形成5-(4-羟苯基)-5-苯乙内酰脲时的对映体过量。
