Protein-ligand coupled motions in DHFR catalysis

酶是高效的催化剂，与非催化反应相比，可以实现高达21个数量级的速率提升。然而，尽管进行了几十年的实验，但这些显著的速率提高的确切原因并不完全清楚。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-底物复合体中的运动。酶运动在催化反应的物理步骤(即底物结合、产物释放和全球构象变化)中扮演的角色已经得到了很好的证实。然而，这种动态运动对酶催化反应实际化学的影响还不是很清楚。尤其是快速运动对反应的积极促进作用是当前机械酶催化研究的热点。我们将利用二氢叶酸还原酶(DHFR)来研究动力学与酶化学之间的关系。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。与生俱来的不可避免的抗药性问题，以及筛选程序的低产量，需要一种合理的方法来开发新的抑制剂，这是基于对催化过程的机理和动态细节的彻底了解。在我们之前广泛研究的基础上，我们将通过以下方式来解决这一问题：1)使用化学和酶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(开发和模拟生化途径)允许对所涉及分子中的特定位置进行有效标记；这种特定标记不是通过传统化学方法容易实现的。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)直接报告局部动态运动，由于标记化合物的制备的复杂性，到目前为止还没有测量到。我们如上所述的策略现在允许直接访问这些化合物。将这些重原子KIE与我们现有的、使用计算模型的全面氢KIE数据相结合，将提供DHFR催化反应的过渡态的详细图。3)蛋白质、底物和辅因子动力学的耦合将通过核磁共振光谱进行研究。结合现代核磁共振技术，对反应的所有组分进行特定的“量身定制”的标记，首次使人们能够彻底调查活性酶复合体中的快速运动对反应的贡献。总的来说，这个项目将提供详细的洞察，了解酶反应中动力学和催化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 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.