Analysis of coumarin 7-hydroxylation activity of cytochrome P450 2A6 using random mutagenesis
Analysis of coumarin 7-hydroxylation activity of cytochrome P450 2A6 using random mutagenesis
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DOI：
10.1074/jbc.m508171200
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发表时间：
2005-12-02
影响因子：
4.8
通讯作者：
Guengerich, FP
中科院分区：
文献类型：
--
作者：
Kim, D;Wu, ZL;Guengerich, FP
细胞色素P450（P450）2A 6是一种重要的人体酶，参与许多异生物质化学物质的代谢，包括香豆素，吲哚，尼古丁和致癌亚硝胺。在P450 2A 6的分析中使用随机诱变和高通量筛选的组合，利用荧光香豆素7-羟基化测定。测定了野生型P450 2A 6和35个选定突变体香豆素7-羟基化的稳态动力学参数（k（cat）和K-m），表明整个编码区的突变体对活性有影响。五个突变体显示降低催化效率（kcat/Km），进一步分析底物的选择性和结合亲和力，并显示7-甲氧基香豆素O-脱甲基，叔丁基甲基醚O-脱甲基，吲哚3-羟基化的催化活性降低。除一种突变体（K476 E）外，所有突变体均表现出香豆素结合亲和力下降（以及较高的Km值），这表明这是酶活性下降的主要基础。结合到香豆素的P450 2A 6的最近的X射线晶体结构（Yano，J.K.，徐，M. H、格里芬，K。J.，斯托特角D、和约翰逊，E. F.（2005）Nat. Struct. Mol. 12，822 - 823）表明，回收的A481 T和N297 S突变似乎接近香豆素，表明底物相互作用的直接扰动。K476 E突变体的酶活性降低与NADPH氧化和铁P450 2A 6-香豆素复合物的还原速率降低有关。减弱部分是由于降低了对NADPH-P450还原酶的结合亲和力，但K476 E突变体即使在高还原酶浓度下也没有达到野生型香豆素7-羟基化活性。
Cytochrome P450 ( P450) 2A6 is an important human enzyme involved in the metabolism of many xenobiotic chemicals including coumarin, indole, nicotine, and carcinogenic nitrosamines. A combination of random mutagenesis and high-throughput screening was used in the analysis of P450 2A6, utilizing a fluorescent coumarin 7-hydroxylation assay. The steady-state kinetic parameters (k(cat) and K-m) for coumarin 7-hydroxylation by wild-type P450 2A6 and 35 selected mutants were measured and indicated that mutants throughout the coding region can have effects on activity. Five mutants showing decreased catalytic efficiency (kcat/Km) were further analyzed for substrate selectivity and binding affinities and showed reduced catalytic activities for 7-methoxycoumarin O-demethylation, tert-butyl methyl ether O-demethylation, and indole 3-hydroxylation. All mutants except one (K476E) showed decreased coumarin binding affinities ( and also higher Km values), indicating that this is a major basis for the decreased enzymatic activities. A recent x-ray crystal structure of P450 2A6 bound to coumarin (Yano, J. K., Hsu, M. H., Griffin, K. J., Stout, C. D., and Johnson, E. F. ( 2005) Nat. Struct. Mol. Biol. 12, 822 - 823) indicates that the recovered A481T and N297S mutations appear to be close to coumarin, suggesting direct perturbation of substrate interaction. The decreased enzymatic activity of the K476E mutant was associated with decreases both in NADPH oxidation and the reduction rate of the ferric P450 2A6-coumarin complex. The attenuation is caused in part to lower binding affinity for NADPH-P450 reductase, but the K476E mutant did not achieve the wild-type coumarin 7-hydroxylation activity even at high reductase concentrations.