Structural Basis of Cytochrome P450 Activity

描述（由申请人提供）：人类细胞色素P450 （P450）酶是膜蛋白，在疏水外来分子的代谢中起关键作用。这种外源代谢使大多数药物失活，但可以将前药和前致癌物分别转化为活性或致癌物。每一种P450酶都可以结合化学上不同的底物，然后接受nadph -细胞色素P450还原酶的一个电子，结合分子氧，最后接受第二个电子使底物单氧化。小血红素蛋白细胞色素b5可以不同程度地加速、抑制或不影响这种催化作用。因此，为了完成药物代谢或前致癌物激活，P450与化学上非常独特的小分子底物和多种蛋白质伴侣相互作用。了解和预测哪些不同的外源化合物被识别，以及它们如何被不同的P450酶代谢，不仅对了解我们所接触的现有药物和其他化学物质的代谢，而且对指导新药的开发和化学预防工作具有重要的实用价值。然而，关于有多少不同的人类P450蛋白导向底物的结构信息，例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，并最终阻碍了对各种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具有临床应用价值的重要人类P450酶的初始结构，检查临床重要的P450/配体复合物，以及探索P450与参与催化的其他蛋白质之间的关系，来扩展我们的结构知识。Aim 1的重点是新的肺1A1和2F酶的x射线结构，这些酶的前致癌物被激活以引发肺癌，此外还有2W1，一种P450，可用于选择性地激活结肠癌治疗的抗癌前药物。这些膜P450酶彼此之间以及与最近研究的2A13和2E1酶共享特定底物，推进了确定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类P450与还原酶和b5蛋白的相互作用，这些复合物目前没有结构可用。P450/还原酶和P450/b5复合物将使用x射线晶体学对选定的人类P450酶进行详细定义，而核磁共振提供了在多种医学相关的人类P450酶之间轻松比较和对比这种相互作用的机会。基于我们目前的数据和人类膜P450酶的结构和功能分析能力，这项工作有望为多种人类酶提供先进的结构/功能信息，这些酶可以直接应用于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.