Structural Basis of Cytochrome P450 Activity

描述（由申请方提供）：人细胞色素P450（P450）酶是在疏水性外源分子代谢中发挥关键作用的膜蛋白。这种异生物质代谢使大多数药物失活，但可将前药和原致癌物分别转化为活性或致癌形式。这些P450酶中的每一种都可以结合化学上不同范围的底物，然后从NADPH-细胞色素P450还原酶接受一个电子，结合分子氧，最后接受第二个电子以单取代底物。小血红素蛋白质细胞色素b5可以加速、抑制或不影响这种催化作用。因此，为了实现药物代谢或前致癌原活化，P450与化学性质非常不同的小分子底物和多种蛋白质伴侣相互作用。了解并能够预测哪些不同的外来化合物被识别，以及它们如何被不同的P450酶代谢，不仅在了解我们所接触的现有药物和其他化学品的代谢方面具有重要的实用价值，而且在指导新药和化学预防工作的开发方面也具有重要的实用价值。然而，关于有多少不同的人类P450蛋白定向底物的结构信息，比如代谢为致癌形式与非致癌形式，以及还原酶和b5蛋白相互作用如何调节这种催化作用是我们知识中的一个空白。这阻碍了潜在有价值的抗癌药物的有效使用，阻碍了对癌症起始事件的理解，并最终阻碍了对不同疾病状态的预防和治疗。申请人的长期研究目标是了解控制P450催化的结构/功能原理，以便利用这些信息来改善人类健康。本提案的目的是通过确定临床实用的重要人类P450酶的初始结构，检查临床重要的P450/配体复合物，并探测P450和其他参与催化的蛋白质之间的关系，来扩展我们的结构知识。目的1聚焦于肺1A 1和2F酶的新X射线结构，其具有活化以引发肺癌的前致癌物，以及2 W1，一种可用于选择性地活化抗癌前药以用于结肠癌治疗的P450。这些膜P450酶在它们之间以及与最近研究的2A 13和2 E1酶共享特定底物，推进了鉴定负责部分重叠但不同代谢的结构特征的长期目标。第二个目标从P450/配体相互作用转移到人P450与还原酶和b5蛋白的相互作用，目前还没有结构的复合物。P450/还原酶和P450/b5复合物将使用X射线晶体学详细定义选择的人P450酶，而NMR提供了容易比较和对比多种医学相关的人P450酶之间的相互作用的机会。基于我们目前的数据和对人类膜P450酶的结构和功能分析的能力，这项工作有望为多种人类酶提供先进的结构/功能信息，这些酶可直接应用于促进多种疾病状态下的人类健康，但重点是肺癌和结肠癌。

DESCRIPTION (provided by applicant): Human cytochrome P450 (P450) enzymes are membrane proteins that play key roles in the metabolism of hydrophobic foreign molecules. Such xenobiotic metabolism deactivates most drugs, but can transform prodrugs and procarcinogens into active or carcinogenic forms, respectively. Each of these P450 enzymes can bind a chemically diverse range of substrates, then accept an electron from NADPH-cytochrome P450 reductase, bind molecular oxygen, and finally accept a second electron to monooxygenate the substrate. The small heme protein cytochrome b5 can variably accelerate, inhibit, or have no effect on this catalysis. Thus to accomplish drug metabolism or procarcinogen activation, a P450 interacts with very chemically distinct small molecule substrates and with multiple protein partners. Understanding and being able to predict which diverse foreign compounds are recognized and how they are metabolized by different P450 enzymes is of great practical value not only in understanding the metabolism of existing drugs and other chemicals to which we are exposed, but also in guiding the development of new drugs and chemoprevention efforts. However, structural information about how many different human P450 proteins orient substrates, say for metabolism to a carcinogenic form vs. a noncarcinogenic form, and how reductase and b5 protein interactions regulate such catalysis is a gap in our knowledge. This prevents the effective use of potentially valuable anticancer agents, understanding of cancer initiation events, and ultimately improved prevention and treatment of diverse disease states. The applicant's long-term research goal is to understand the structure/function principles that control P450 catalysis, in order that this information can b exploited to improve human health. The objective of this proposal is to extend our structural knowledge across current boundaries by determining initial structures for important human P450 enzymes of clinical utility, examining clinically-important P450/ligand complexes, and probing the relationships between P450 and other proteins involved in catalysis. Aim 1 focuses on new X-ray structures of lung 1A1 and 2F enzymes with procarcinogens activated to initiate lung cancer, in addition to 2W1, a P450 that can be used to activate anticancer prodrugs selectively for colon cancer treatment. These membrane P450 enzymes share specific substrates among themselves and with the 2A13 and 2E1 enzymes recently studied, advancing the longer-term goal of identifying structural features responsible for partially overlapping, yet distinct metabolism. The second aim moves from P450/ligand interactions to human P450 interactions with reductase and b5 proteins, complexes for which no structures are currently available. P450/reductase and P450/b5 complexes will be defined in detail for select human P450 enzymes using X-ray crystallography, while NMR provides the opportunity to readily compare and contrast such interactions across multiple medically-relevant human P450 enzymes. Based on our current data and demonstrated capabilities in structural and functional analysis of human membrane P450 enzymes, this work is expected to provide advanced structure/function information for multiple human enzymes that can be directly applied to advance human health in multiple disease states, but with a focus on lung and colon cancers.