Reaction-coupled dynamics in DHFR catalysis

酶是高效的催化剂，与非催化反应相比，速度提高高达21个数量级。然而，这些显著的速率提高的确切原因还不完全清楚。氢转移反应在所有生物过程中都是至关重要的。为了了解控制这些反应速度的影响，必须考虑酶的运动。尤其是积极促进反应的快速运动的影响，是目前酶催化机理研究的热点。酶是大分子，但我们已经证明，虽然长程酶运动在催化循环的物理步骤(即底物结合、产物释放和全球构象变化)中发挥重要作用，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、局部的酶运动确实在化学步骤中发挥了作用，但这种作用并不是传统上提出的。现在我们对酶运动与化学步骤的耦合速度有了更彻底的总体了解，我们能够集中精力对所涉及的运动进行精确的原子学理解。我们将使用二氢叶酸还原酶(DHFR)来研究动力学和酶化学之间的关系。这种酶在许多重要的生化过程中是必需的，包括DNA和氨基酸的合成。因此，它是一个长期确立的药物靶点，已经发现并成功地开发了几种抑制剂作为抗菌、抗疟疾和抗肿瘤药物。耐药性的增加和不可避免的问题，以及筛选方案的低产量，要求在彻底了解催化过程的机理和动态细节的基础上，采取合理的方法来开发新的抑制剂。基于我们之前广泛的研究，我们将通过以下方式来解决这个问题：-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一个强有力的策略，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；现在我们寻求确定所涉及的酶的特定部分。我们可以用细菌培养方法生产标记或未标记的蛋白质的个别部分，并将不同区域化学连接在一起，形成全长的有活性的酶。或者，我们可以通过向培养物喂入标记的氨基酸或其生化前体来直接结合标记。-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。我们已经证明，酶的完全标记对动力学有显著的影响。然而，很可能只有酶的某些部分才会引起这种影响。通过将各种选择性标记的模式与完全标记的酶的结果进行比较，我们将准确地确定直接涉及的区域。-研究选择性同位素标记对酶动力学的影响。通过在感兴趣的位置加入特定的标记，并在其他位置通过随机分段标记改变酶的总质量，我们可以使用磁共振技术确定对酶动力学的影响。这将是对动力学研究的补充，并将提供对快速运动在活性酶复合体中的贡献的彻底调查。总体而言，这个项目将提供详细的见解，了解动力学和催化在酶反应中是如何联系在一起的。它最终将使我们能够开发出一种催化模型，能够解释大自然催化剂的巨大效率，并应导致酶抑制剂的合理设计，将其应用于抗感染和抗癌药物。

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.