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Protein-ligand coupled motions in DHFR catalysis

DHFR 催化中的蛋白质-配体耦合运动

基本信息

批准号：
BB/J005266/1
负责人：
Rudolf Allemann
金额：
$ 54.69万
依托单位：
CARDIFF UNIVERSITY
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2012
资助国家：
英国
起止时间：
2012 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FJ005266%2F1
关键词：
Protein ligand coupled motions DHFR

项目摘要

Enzymes are efficient catalysts that can achieve rate enhancements of up to 21 orders of magnitude relative to the uncatalysed reactions. However, despite many decades of experimentation, the precise causes of these remarkable rate enhancements are not fully understood. Hydrogen transfer reactions are of fundamental importance in all biological processes. In order to understand the effects that control the speed of these reactions, motions in the enzyme-substrate complex must be taken into account. The role that enzyme motions play in the physical steps of the catalysed reaction (i.e. binding of substrates, release of products and global conformational changes) is well established. However, the influence of such dynamic motions on the actual chemistry of an enzyme-catalysed reaction is less well defined. In particular, the influence of fast motions that actively promote the reaction is a current hot topic in mechanistic enzyme catalysis.We will investigate the correlation between dynamics and enzymatic chemistry using the enzyme dihydrofolate reductase (DHFR). This enzyme is required in many essential biochemical processes including the synthesis of DNA and amino acids. It is therefore a long established drug target and several inhibitors have been discovered and successfully developed as antibacterial, antimalarial and anti-tumour drugs. The increasing and inherently unavoidable problem of drug resistance together with the poor yield from screening programmes demands a rational approach to develop new inhibitors based on a thorough understanding of the mechanistic and dynamic details of the catalytic process.Based on our extensive previous research, we will approach this in the following way:1) Isotopic labelling of the reactant molecules using chemical and enzymatic processes. Especially our multi-enzyme syntheses (exploiting and mimicking biochemical pathways) allow the efficient labelling of specific positions in the molecules involved; such specific labelling is no easily achieved by conventional chemical methods. These isotope-labelled compounds are essential for the investigations described under 2) and 3).2) Heavy atom kinetic isotope effects (KIE of carbon and nitrogen in this case), which report directly on local dynamic motions, have not been measured to date due to the complexity of preparation of the labelled compounds. Our strategy described above now allows straightforward access to these compounds. A combination of these heavy atom KIEs with our existing, comprehensive hydrogen KIE data using computational models will provide a detailed map of the transition state of the DHFR catalysed reaction.3) The coupling of the dynamics of protein, substrate and cofactor will be investigated by nuclear magnetic resonance spectroscopy. The specific, 'tailor-made' labelling of all components of the reaction in combination with modern NMR techniques allows for the first time a thorough investigation of the contributions of fast motions in the active enzyme complex on the reaction.Overall, this project will provide detailed insight into how dynamics and catalysis are linked in enzymatic reactions. It will eventually allow us to develop a model of catalysis that can explain the enormous efficiency of Nature's catalysts and should lead to the rational design of enzyme inhibitors with applications as anti-infective and anti-cancer agents.

酶是有效的催化剂，相对于未催化的反应，可以实现高达21个数量级的速率提高。然而，尽管经过几十年的实验，这些显着的速率提高的确切原因尚未完全理解。氢转移反应在所有生物过程中具有根本的重要性。为了理解控制这些反应速度的效应，必须考虑酶-底物复合物中的运动。酶运动在催化反应的物理步骤（即底物的结合、产物的释放和全局构象变化）中所起的作用是公认的。然而，这种动态运动对酶催化反应的实际化学性质的影响还不太清楚。特别是，快速运动的影响，积极促进反应是当前的热点话题在机械酶催化。我们将调查动力学和酶化学之间的相关性使用酶二氢叶酸还原酶（DHFR）。这种酶在许多基本的生物化学过程中是必需的，包括DNA和氨基酸的合成。因此，它是一个长期建立的药物靶标，并且已经发现并成功开发了几种抑制剂作为抗菌，抗疟疾和抗肿瘤药物。随着药物耐药性问题的日益严重，以及药物筛选过程中的低产量，我们需要在深入了解催化过程的机理和动力学细节的基础上，采用合理的方法来开发新的抑制剂。基于我们以前的广泛研究，我们将采用以下方法：1）使用化学和酶促过程对反应物分子进行同位素标记。特别是我们的多酶合成（利用和模拟生物化学途径）允许有效标记所涉及分子中的特定位置;这种特异性标记不容易通过常规化学方法实现。这些同位素标记的化合物对于2）和3）中描述的研究是必不可少的。2）重原子动力学同位素效应（在这种情况下是碳和氮的KIE），其直接报告局部动力学运动，由于标记化合物的制备的复杂性，迄今尚未测量。我们的上述策略现在允许直接获得这些化合物。结合这些重原子KIE与我们现有的，全面的氢KIE数据使用计算模型将提供一个详细的地图的过渡态的DHFR催化反应。3）耦合的蛋白质，底物和辅因子的动力学将通过核磁共振光谱研究。结合现代核磁共振技术，对反应的所有组分进行特定的“定制”标记，首次彻底研究了活性酶复合物中快速运动对反应的贡献。总体而言，该项目将详细了解酶促反应中动力学和催化作用的联系。它最终将使我们能够开发一种催化模型，可以解释自然界催化剂的巨大效率，并应导致酶抑制剂的合理设计，作为抗感染和抗癌剂的应用。