Phenylalanine and tryptophan scanning mutagenesis of CYP3A4 substrate recognition site residues and effect on substrate oxidation and cooperativity
Phenylalanine and tryptophan scanning mutagenesis of CYP3A4 substrate recognition site residues and effect on substrate oxidation and cooperativity
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DOI：
10.1021/bi010758a
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发表时间：
2001-08-28
期刊：
影响因子：
2.9
通讯作者：
Halpert, JR
中科院分区：
文献类型：
--
作者：
Domanski, TL;He, YA;Halpert, JR
在CYP 3A 4内的15个位点进行苯丙氨酸和/或色氨酸扫描诱变，这些位点可能与底物特异性或协同性有关。这些位点是根据以前的研究或与含有两个雄烯二酮分子的P450（eryF）的结构进行比较而选择的。在使用孕酮、睾酮、7-苄氧基-4-（三氟甲基）香豆素（7-BFC）和α-萘甲酮（ANF）作为底物的重构系统中评估25种突变体的功能。CYP 3A 4野生型显示ANF 5，6-氧化物形成和7-BFC脱苄基的S形动力学。对12个具有显著类固醇羟化酶活性的突变体的分析显示，ANF氧化和ANF对孕酮6 β-羟化的刺激之间缺乏正相关性，表明ANF结合在CYP 3A 4内的两个位点。7-孕酮和心钠素可刺激BFC脱苄基化，而7-BFC不抑制睾酮或孕酮6 β-羟基化.相关性分析显示7-BFC脱苄基化与孕酮或睾酮6 β-羟基化之间无相关性。这些数据很难用CYP 3A 4的双位点模型来解释，但表明活性位点内存在三个亚口袋。有趣的是，根据其氧化本研究中使用的四种底物的能力对突变体进行分类，表明底物确实在CYP 3A 4结合口袋中的优选位置结合。
Phenylalanine and/or tryptophan scanning mutagenesis was performed at 15 sites within CYP3A4 proposed to be involved in substrate specificity or cooperativity. The sites were chosen on the basis of previous studies or from a comparison with the structure of P450(eryF) containing two molecules of androstenedione. The function of the 25 mutants was assessed in a reconstituted system using progesterone, testosterone, 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC), and alpha -naphthoflavone (ANF) as substrates. CYP3A4 wild type displayed sigmoidal kinetics of ANF 5,6-oxide formation and 7-BFC debenzylation. Analysis of 12 mutants with significant steroid hydroxylase activity showed a lack of positive correlation between ANF oxidation and stimulation of progesterone 6 beta -hydroxylation by ANF, indicating that ANF binds at two sites within CYP3A4. 7-BFC debenzylation was stimulated by progesterone and ANF, and 7-BFC did not inhibit testosterone or progesterone 6 beta -hydroxylation. Correlational analysis showed no relationship between 7-BFC debenzylation and either progesterone or testosterone 6 beta -hydroxylation. These data are difficult to explain with a two-site model of CYP3A4 but suggest that three subpockets exist within the active site. Interestingly, classification of the mutants according to their ability to oxidize the four substrates utilized in this study suggested that substrates do bind at preferred locations in the CYP3A4 binding pocket.