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Novel routes to catalytic intermediates in the cytochrome P450 catalytic cycle

细胞色素 P450 催化循环中催化中间体的新途径

基本信息

批准号：
BB/F002521/1
负责人：
Andrew Munro
金额：
$ 67.71万
依托单位：
University of Manchester
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2007
资助国家：
英国
起止时间：
2007 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FF002521%2F1
关键词：
Novel routes catalytic intermediates cytochrome

项目摘要

The proteins known as cytochromes P450 (P450s) are essential in physiology of all life forms. They are heme-binding proteins, and bind the same heme cofactor as does the oxygen carrying blood protein hemoglobin. Like hemoglobin, P450s also bind molecular oxygen (O2). However, unlike hemoglobin they reduce bound oxygen with electrons delivered to the heme from partner proteins, and which ultimately are derived from the cell coenzyme NADPH. This enables P450s to split the oxygen molecule into its component atoms. One of the two atoms is used to form water (H2O), while the other is used to oxygenate an organic substrate molecule bound by the P450 close to its heme iron. Frequently, hydroxylation (introduction of an OH group) is catalysed. In humans, activity of P450s is essential for production of steroid hormones, and also for creation of many lipid molecules essential for signalling within the body (e.g. for activation of the immune system). However, humans have 57 different P450s, and their most famous roles are in detoxification and removal of drugs and other xenobiotics from the body / performed mainly by hepatic P450s. In bacteria and lower eukaryotes, the P450s have important roles in pathways that allow unusual molecules (e.g. camphor) to be used to provide energy for growth, and are essential for production of molecules such as antibiotics (e.g. erythromycin). The ability of P450 enzymes to introduce oxygen atoms at defined positions in organic molecules has also attracted much attention from organic chemists, who are looking for cleaner and more environmentally friendly routes to synthesis of drugs and other important molecules. A fundamental understanding of P450 structure and activity is essential to understand how they achieve their biological functions, and how they can be applied for biotechnological roles. Also, there is enormous interest in understanding how prescribed drugs bind to individual P450s (and how molecules of biotechnological interest bind to the relevant P450s), since this can lead to accurate predictions of how individual P450s act on these molecules, their lifetimes in the body and how these parameters can be changed by altering the drug structure. The usual way of determining binding modes of substrates/drugs to P450s is to form crystals of the complex made between the P450 and the drug, and then use the technique of x-ray diffraction to obtain the crystal structure. In this proposal, we seek to address fundamental questions relating to how P450s 'activate' oxygen and catalyse hydroxylation reactions. Specifically, we will use modern kinetic techniques (including laser flash photolysis) to provide evidence for formation of transient reactive heme species that are considered critical for oxygenation chemistry. Also, we will use these methods to answer a critical question relating to whether two different reactive species are formed in the P450 reaction 'cycle' and if these have differing types of activities that could be exploited biotechnologically. In addition, we will address serious issues relating to the relevance of binding modes seen for substrates in different P450 x-ray structures. We will use a model system (P450 BM3) to establish whether an observed substrate binding mode is relevant to catalysis in the P450 and to challenge hypotheses suggesting that the substrate re-positions as the P450 is reduced, or whether thermal effects are critical for causing substrate to relocate. Collectively, this work will answer fundamental questions on the nature of P450 catalysis and the relevance of distinct reactive intermediates in the process. Also, it will define the relevance of substrate binding mode and substrate relocation in a key model P450, with important ramifications for rationalising how substrates bind to biomedically relevant P450s. Thus, the study proposed has wide ranging relevance to understanding P450 activity in mammalian physiology and for biotechnological applications.

细胞色素 P450 (P450s) 蛋白质对于所有生命形式的生理学至关重要。它们是血红素结合蛋白，与携氧血液蛋白血红蛋白结合相同的血红素辅因子。与血红蛋白一样，P450 也结合分子氧 (O2)。然而，与血红蛋白不同的是，它们通过从伙伴蛋白传递到血红素的电子来减少结合氧，这些电子最终源自细胞辅酶 NADPH。这使得 P450 能够将氧分子分裂成其组成原子。两个原子中的一个用于形成水 (H2O)，而另一个则用于氧化由接近其血红素铁的 P450 结合的有机底物分子。通常，羟基化（引入 OH 基团）会被催化。在人类中，P450 的活性对于类固醇激素的产生以及体内信号传导（例如激活免疫系统）所必需的许多脂质分子的产生至关重要。然而，人类有 57 种不同的 P450，它们最著名的作用是解毒和清除体内的药物和其他异生物质/主要由肝脏 P450 执行。在细菌和低等真核生物中，P450 在允许使用不寻常分子（例如樟脑）为生长提供能量的途径中发挥重要作用，并且对于抗生素（例如红霉素）等分子的生产至关重要。 P450酶在有机分子的特定位置引入氧原子的能力也引起了有机化学家的广泛关注，他们正在寻找更清洁、更环保的途径来合成药物和其他重要分子。对 P450 结构和活性的基本了解对于了解它们如何实现其生物功能以及如何将它们应用于生物技术角色至关重要。此外，人们对了解处方药物如何与单个 P450 结合（以及生物技术感兴趣的分子如何与相关 P450 结合）非常感兴趣，因为这可以准确预测单个 P450 如何作用于这些分子、它们在体内的寿命以及如何通过改变药物结构来改变这些参数。确定底物/药物与P450的结合模式的常用方法是形成P450与药物之间的复合物的晶体，然后利用X射线衍射技术获得晶体结构。在本提案中，我们寻求解决有关 P450 如何“激活”氧气并催化羟基化反应的基本问题。具体来说，我们将使用现代动力学技术（包括激光闪光光解）来提供瞬时反应性血红素物质形成的证据，这些物质被认为对氧化化学至关重要。此外，我们将使用这些方法来回答一个关键问题，即 P450 反应“循环”中是否形成两种不同的活性物质，以及它们是否具有可以通过生物技术利用的不同类型的活性。此外，我们将解决与不同 P450 X 射线结构中的底物结合模式相关性相关的严重问题。我们将使用模型系统 (P450 BM3) 来确定观察到的底物结合模式是否与 P450 中的催化相关，并挑战表明底物随着 P450 减少而重新定位的假设，或者热效应是否对于导致底物重新定位至关重要。总的来说，这项工作将回答有关 P450 催化性质以及该过程中不同反应中间体的相关性的基本问题。此外，它将定义关键模型 P450 中底物结合模式和底物重定位的相关性，对于合理化底物与生物医学相关 P450 的结合方式具有重要影响。因此，这项研究与了解哺乳动物生理学中的 P450 活性和生物技术应用具有广泛的相关性。