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Reaction-coupled dynamics in DHFR catalysis

DHFR 催化中的反应耦合动力学

基本信息

批准号：
BB/L020394/1
负责人：
Rudolf Allemann
金额：
$ 51.33万
依托单位：
CARDIFF UNIVERSITY
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2014
资助国家：
英国
起止时间：
2014 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FL020394%2F1
关键词：
Reaction coupled dynamics DHFR catalysis

项目摘要

Enzymes are efficient catalysts that achieve rate enhancements of up to 21 orders of magnitude relative to uncatalysed reactions. However, 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 controlling the speed of these reactions, enzyme motions must be taken into account. In particular, the influence of fast motions that actively promote the reaction is a current hot topic in mechanistic studies of enzyme catalysis. Enzymes are large molecules, but we have shown that while long-range enzyme motions play important roles in the physical steps of the catalytic cycle (i.e. binding of substrates, release of products and global conformational changes), they have no effect on the actual chemical step. Our recent results have shown that fast, localised enzyme motions do play a role in the chemical step, but that this role is not the one traditionally proposed.Now that we have a more thorough general understanding of how fast enzyme motions couple to the chemical step, we are able to focus our efforts towards a precise atomistic understanding of the motions involved. We will investigate the relationship between dynamics and enzymatic chemistry using the enzyme dihydrofolate reductase (DHFR). This enzyme is required in many essential biochemical processes including 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: - Selective isotopic labelling of the enzyme. Isotopic labelling is a powerful strategy for investigating the role of enzyme dynamics, as the dynamics are affected but other properties of the enzyme are not. We already have data for the fully labelled 'heavy' enzyme; now we seek to identify the specific portions of the enzyme involved. We can produce individual parts of the protein either labelled or unlabelled using bacterial culture methods, and chemically join the different regions together to form the full length, active enzyme. Alternatively, we can incorporate labels directly by feeding the culture with labelled amino acids or their biochemical precursors.- Measuring the effect of selective isotopic labelling on the kinetics of the chemical reaction catalysed by the enzyme. We have shown that full labelling of the enzyme has a significant effect on the kinetics. However, it is likely that only certain parts of the enzyme cause this effect. By comparing various patterns of selective labelling against the results for the fully labelled enzyme, we will pinpoint the regions directly involved.- Investigation of the effect of selective isotopic labelling on the dynamics of the enzyme. By incorporating specific labels at positions of interest, and varying the overall mass of the enzyme by random fractional labelling at other sites, we can determine the effect on the enzyme dynamics using magnetic resonance techniques. This will complement the kinetic studies and will provide a thorough investigation of the contributions of fast motions in the active enzyme complex. 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，其实现高达21个数量级的速率增强。然而，这些显着的速率增强的确切原因还没有完全理解。氢转移反应在所有生物过程中是至关重要的。为了理解控制这些反应速度的效应，必须考虑酶的运动。特别是，快速运动的影响，积极促进反应是当前的热点问题，在酶催化机理的研究。酶是大分子，但我们已经表明，虽然长距离酶运动在催化循环的物理步骤（即底物的结合，产物的释放和全局构象变化）中起着重要作用，但它们对实际的化学步骤没有影响。我们最近的研究结果表明，快速，本地化的酶运动确实在化学步骤中发挥作用，但这种作用是不是一个传统的建议，现在，我们有一个更全面的了解如何快速酶运动耦合到化学步骤，我们能够集中我们的努力，对所涉及的运动精确的原子理解。我们将使用二氢叶酸还原酶（DHFR）研究动力学和酶化学之间的关系。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。耐药性的增加和内在不可避免的问题，以及从筛选方案的产量差，需要一个合理的方法来开发新的抑制剂的基础上彻底了解的催化过程的机制和动态的细节。基于我们以前广泛的研究，我们将以以下方式处理：-酶的选择性同位素标记。同位素标记是研究酶动力学作用的有力策略，因为动力学受到影响，但酶的其他性质不受影响。我们已经有了完全标记的“重”酶的数据;现在我们试图确定所涉及的酶的特定部分。我们可以使用细菌培养方法生产标记或未标记的蛋白质的各个部分，并将不同区域化学连接在一起以形成全长活性酶。或者，我们可以通过用标记的氨基酸或其生化前体喂养培养物来直接掺入标记。测量选择性同位素标记对酶催化的化学反应动力学的影响。我们已经表明，完全标记的酶的动力学有显着的影响。然而，很可能只有酶的某些部分引起这种效应。通过比较选择性标记的各种模式与完全标记酶的结果，我们将查明直接涉及的区域。选择性同位素标记对酶动力学影响的研究。通过在感兴趣的位置加入特定的标签，并通过在其他位点随机分数标记来改变酶的总质量，我们可以使用磁共振技术来确定对酶动力学的影响。这将补充动力学研究，并将提供一个彻底的调查的贡献，快速运动的活性酶复合物。总的来说，这个项目将提供详细的洞察动力学和催化是如何在酶促反应中联系起来的。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。