A 3-DIMENSIONAL MODEL OF AROMATASE CYTOCHROME-P450
A 3-DIMENSIONAL MODEL OF AROMATASE CYTOCHROME-P450
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DOI：
10.1002/pro.5560040605
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发表时间：
1995-06-01
期刊：
影响因子：
8
通讯作者：
SIMPSON, ER
中科院分区：
文献类型：
--
作者：
GRAHAMLORENCE, S;AMARNEH, B;SIMPSON, ER
P450 血红素蛋白包含一个大型基因超家族，在氧化还原伴侣存在的情况下催化单加氧酶反应。由于哺乳动物成员无一例外都是膜结合蛋白，因此它们无法通过 X 射线晶体学方法进行结构功能分析。在 P450 催化的反应中，催化 C19 类固醇转化为雌激素的芳香酶反应是最复杂且了解最少的反应之一。因此，为了更好地理解反应机理，我们构建了 P450arom 的三维模型，不仅可以检查活性位点和可能参与催化的残基，还可以研究其他重要的结构特征，例如底物识别和氧化还原伙伴结合，这需要检查整个分子（除了假定的跨膜区域）。这个 P450arom 模型是基于从可溶性细菌 P450（P450cam、P450terp 和 P450BM-P）的结构中识别出的“核心结构”而构建的，而不是通过分子替换，然后以合理的方式添加不太保守的元件和环。采用最小化和动态仿真对模型进行优化，并评价结构的合理性。根据该模型，我们假设了参与底物识别的脂肪族和芳香族残基的膜相关疏水区域、与其他 P450 相比可能独特的氧化还原伙伴结合区域，以及参与底物活性位点定向的残基和 P450arom 抑制剂（即伏罗唑）。我们还提出了一种反应机制方案，其中“苏氨酸开关”决定氧插入底物分子是否涉及氧自由基或过氧化物中间体。
P450 hemeproteins comprise a large gene superfamily that catalyzes monooxygenase reactions in the presence of a redox partner. Because the mammalian members are, without exception, membrane-bound proteins, they have resisted structure-function analysis by means of X-ray crystallographic methods. Among P450-catalyzed reactions, the aromatase reaction that catalyzes the conversion of C19 steroids to estrogens is one of the most complex and least understood. Thus, to better understand the reaction mechanism, we have constructed a three-dimensional model of P450arom not only to examine the active site and those residues potentially involved in catalysis, but to study other important structural features such as substrate recognition and redox-partner binding, which require examination of the entire molecule (excepting the putative membrane-spanning region). This model of P450arom was built based on a ''core structure'' identified from the structures of the soluble, bacterial P450s (P450cam, P450terp, and P450BM-P) rather than by molecular replacement, after which the less conserved elements and loops were added in a rational fashion. Minimization and dynamic simulations were used to optimize the model and the reasonableness of the structure was evaluated. From this model we have postulated a membrane-associated hydrophobic region of aliphatic and aromatic residues involved in substrate recognition, a redox-partner binding region that may be unique compared to other P450s, as well as residues involved in active site orientation of substrates and an inhibitor of P450arom, namely vorozole. We also have proposed a scheme for the reaction mechanism in which a ''threonine switch'' determines whether oxygen insertion into the substrate molecule involves an oxygen radical or a peroxide intermediate.