Cooperativity in oxidations catalyzed by cytochrome P450 3A4
Cooperativity in oxidations catalyzed by cytochrome P450 3A4
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DOI：
10.1021/bi962359z
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发表时间：
1997-01-14
期刊：
影响因子：
2.9
通讯作者：
Guengerich, FP
中科院分区：
文献类型：
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作者：
Ueng, YF;Kuwabara, T;Guengerich, FP
细胞色素P450(P450)3A4是人类中含量最丰富的P450，可氧化多种底物，包括各种药物、类固醇、致癌物和大环内酯类天然产物。在一些反应中，在微粒体研究中已经报道了正的协同作用。黄酮类化合物，例如7，8-苯并黄酮(α-萘黄酮)。阿尔法核因子)，已被证明可以刺激一些反应，但不会刺激其他反应。在含有纯化的重组细菌P450 3A4的系统中，几种底物的氧化反应显示出正的协同性，包括睾酮、17β-雌二醇、阿米替林，最显著的是黄曲霉毒素(AF)B-1。在这些反应和其他反应中，阿尔法核因子通常会降低协作性(即Hill图中的n值)，同时刺激或抑制反应。以AFB(1)为底物，α-核黄素既能促进8，9-环氧化反应，又能抑制3-α-羟化反应。在融合的P450 3A4-NADPH-P450还原酶蛋白中，AFB(1)也出现了相同的图谱。在含有单个蛋白质的系统中，阿尔法核糖核酸不会改变作为NADPH-P450还原酶浓度函数的活性模式。在NADPH-P450还原酶被黄还蛋白或铁氧还蛋白、碘代苯或过氧化氢异丙苯取代的体系中，AFB(1)氧化成两种产物的模式被相当大地改变。与AFB(1)不同的是，AFB(2)不被P450 3A4氧化，但能抑制AFB(1)的氧化。这些和其他结果是在几个可能的模型的背景下考虑的。结果支持一个模型，其中涉及变构中心，尽管这个假定的地点与催化中心的接近程度不能被确定为VET。为了解释α-纳豆素和还原体系对黄曲霉毒素B(1)两种氧化作用的不同影响，提出了一个模型，在该模型中，特定构象的底物结合可以促进氧的活化以增强催化作用。
Cytochrome P450 (P450) 3A4 is the most abundant human P450 and oxidizes a diversity of substrates, including various drugs, steroids, carcinogens, and macrolide natural products. In some reactions, positive cooperativity has been reported in microsomal studies. Flavonoids, e.g., 7,8-benzoflavone (alpha-naphthoflavone. alpha NF), have been shown to stimulate some reactions but not others. In systems containing purified recombinant bacterial P450 3A4, positive cooperativity was seen in oxidations of several substrates, including testosterone, 17 beta-estradiol, amitriptyline, and most notably aflatoxin (AF) B-1. With these and other reactions, alpha NF typically reduced cooperativity (i.e., the n value in a Hill plot) while either stimulating or inhibiting reactions. With the substrate AFB(1), alpha NF both stimulated 8,9-epoxidation and inhibited 3 alpha-hydroxylation. The same patterns were seen with AFB(1) in a fused P450 3A4-NADPH-P450 reductase protein. alpha NF did not alter patterns of activity plotted as a function of NADPH-P450 reductase concentration in systems containing the individual proteins. The patterns of AFB(1) oxidation to the two products were modified considerably in systems in which NADPH-P450 reductase was replaced with a flavodoxin or ferredoxin system, iodosylbenzene, or cumene hydroperoxide. AFB(2), which differs from AFB(1) only in the presence of a saturated 8,9-bond, was not oxidized by P450 3A4 but could inhibit AFB(1) oxidation. These and other results are considered in the context of several possible models. The results support a model in which an allosteric site is involved, although the proximity of this putative site to the catalytic site cannot be ascertained as of vet. In order to explain the differential effects of alpha NF and reduction systems on the two oxidations of AFB(1), a model is presented in which binding of substrate in a particular conformation can facilitate oxygen activation to enhance catalysis.