Fowler Stephen M, Taylor John M, Friedberg Thomas, Wolf C Roland, Riley Robert J
Physical and Metabolic Science, AstraZeneca R&D Charnwood, Loughborough, United Kingdom.
Drug Metab Dispos. 2002 Apr;30(4):452-6. doi: 10.1124/dmd.30.4.452.
The leucine 211 --> phenylalanine (L211F) and leucine 211 --> tyrosine (L211Y) mutant forms of cytochrome P450 3A4 have been generated by site-directed mutagenesis and expressed functionally in Escherichia coli. Substrate binding affinities (S50 values) for testosterone and 7-benzyloxy-4-trifluoromethylcoumarin (BFC) were similar for the mutants and wild-type CYP3A4 (49 and 21 microM for L211F, 35 and 20 microM for L211Y, and 33 and 20 microM for the wild type, respectively). For erythromycin, however, the K(m) values determined for the L211F and L211Y mutants were 2.4- and 10.5-fold higher than for the wild type. Furthermore, IC50 values for the inhibition of testosterone 6 beta-hydroxylation by erythromycin and troleandomycin for L211F were 2.4- and 3.7-fold higher, and those for L211Y were 3.4- and 9.2-fold higher than those measured for the wild type. Conversely, small inhibitors, such as diazepam, exhibited no significant difference in IC50 values between the wild type and the L211F and L211Y mutants. It is proposed that large substrates bound in the catalytic center of CYP3A4 with molecular volumes greater than approximately 600 A(3) were less well accommodated in the altered active sites, resulting in lower association energies and increased IC50 values.
通过定点诱变产生了细胞色素P450 3A4的亮氨酸211→苯丙氨酸（L211F）和亮氨酸211→酪氨酸（L211Y）突变形式，并在大肠杆菌中进行了功能表达。突变体和野生型CYP3A4对睾酮和7-苄氧基-4-三氟甲基香豆素（BFC）的底物结合亲和力（S50值）相似（L211F分别为49和21μM，L211Y分别为35和20μM，野生型分别为33和20μM）。然而，对于红霉素，L211F和L211Y突变体的K(m)值比野生型高2.4倍和10.5倍。此外，红霉素和醋竹桃霉素对L211F抑制睾酮6β-羟基化的IC50值分别比野生型高2.4倍和3.7倍，L211Y的则高3.4倍和9.2倍。相反，地西泮等小分子抑制剂在野生型与L211F和L211Y突变体之间的IC50值没有显著差异。有人提出，分子体积大于约600 Å(3)的大底物在CYP3A4催化中心结合时，在改变的活性位点中容纳性较差，导致结合能降低和IC50值增加。
