Structural Basis of Cytochrome P450 Activity

描述(申请人提供)：人细胞色素P450(P450)酶是膜蛋白，在疏水性外来分子的新陈代谢中起关键作用。这种异物代谢使大多数药物失去活性，但可以将前药物和前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一系列不同的底物，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来单氧化底物。小的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的激活，P450与化学上截然不同的小分子底物和多个蛋白质伙伴相互作用。了解和预测哪些不同的外来化合物被识别，以及它们是如何被不同的P450酶代谢的，这不仅对于了解我们接触到的现有药物和其他化学品的代谢，而且对于指导新药的开发和化学预防工作都具有重要的实用价值。然而，关于有多少不同的人类P450蛋白定位底物的结构信息，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，阻碍了对癌症启动事件的了解，并最终改善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。这个建议的目的是通过确定临床上重要的人类P450酶的初始结构，检查临床上重要的P450/配体复合体，以及探索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重点研究肺1A1和2F酶的新的X射线结构，其中包括2W1，2W1是一种可选择性激活抗癌前体药物用于结肠癌治疗的P450。这些膜P450酶彼此共享特定的底物，并与最近研究的2A13和2E1酶共享特定底物，从而推进了识别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，目前还没有可用的结构。将使用X射线结晶学为选定的人类P450酶详细定义P450/还原酶和P450/b5复合体，而核磁共振则提供了容易地比较和对比多种医学相关的人类P450酶的这种相互作用的机会。基于我们目前的数据和对人膜P450酶结构和功能的分析能力，这项工作有望为多种人类酶提供先进的结构/功能信息，这些酶可以在多种疾病状态下直接应用于促进人类健康，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.