The Role of Dynamics in Enzyme Mechanism and Inhibition

描述(申请人提供)：酶是一种非凡的纳米机器，可以驱动细胞过程。研究它们的基本性质是为了更好地了解催化作用，研究它们是为了提高我们设计针对它们的疗法的能力，以造福人类健康。在对酶进行了几十年的结构生物学研究后，我们对它们如何工作的了解仍在增长。最近的工作，特别是在核磁共振光谱学领域，已经揭示了动态运动和预先存在的构象转换对于许多酶的功能是关键的，如果不是所有的话。在这项提案中，最近发展起来的大蛋白质核磁共振自旋弛豫研究方法，结合小分子合成和稳态动力学，将应用于来自大肠杆菌的KD酶-胸苷合成酶(TS)。TS甲基化22-脱氧尿苷52-单磷酸(DUMP)生成22-脱氧胸苷52-单磷酸(DTMP)，从细菌到人类高度保守。胸腺嘧啶核苷的生物合成是一个关键的代谢步骤，如果被抑制，会导致细胞死亡。因此，TS是抗癌药物的靶点，是抗微生物药物的有效靶点。TS是一种专有的同源二聚体，具有复杂的多步骤反应机制，并表现出负的协同性。参与细胞新陈代谢的酶通常具有复杂的机制特征，然而这些分子如何穿越复杂的能量景观来实现其功能还不是很清楚。目前，代谢酶如TS还没有受到其内部构象动力学的彻底分析，这主要是由于它们的大小。因此，TS的特征代表了详细研究大型代谢酶功能运动的早期例子。TS的机制可分解为7个不同的步骤。中间体可以通过底物修饰和突变的特定组合来捕获，这些中间体将被研究其沿反应坐标的动态性质。该方法的一个关键组成部分将不仅是监测蛋白质，而且还监测适当标记的核磁共振弛豫底物的运动。使用小分子作为探针将允许以有效的方式检查所有被捕获的中间体的活性位置的运动。在目标1中，将使用主干和基于甲基的核磁共振弛豫来跟踪底物结合和成键/断键对整个TS的S-ms和PS-ns运动的影响。在目标2中，将从动态和稳态动力学角度考察TS中负协作性的基础。这种方法的关键是混合不对称二聚体的产生。在目标3中，将扩展以前关于二氢叶酸还原酶(DHFR)的发现，以探索抑制剂解离的具体机制(和结构)。将采用一种新的松弛分散方法，以提高对 调节配基解离的运动。我们对酶功能的了解是建立在对小分子酶动力学研究的基础上的。因此，需要一种类似的方法来研究动力学在更大、更复杂的酶中的作用，这应该会在药物设计和蛋白质工程中带来新的策略。

DESCRIPTION (provided by applicant): Enzymes are remarkable nanomachines that drive cellular processes. They are studied for their fundamental properties to better understand catalysis, and they are studied to improve our ability to design therapeutics against them to benefit human health. After decades of structural biology on enzymes, our understanding of how they work is still growing. Recent work, particularly in the area of NMR spectroscopy, for example, has revealed that dynamic motions and pre-existing conformational switching are critical to the functioning of many, if not all, enzymes. In this proposal, recently developed methods for NMR spin relaxation studies on large proteins, paired with small molecule synthesis and steady-state kinetics, will be applied to a 64 kD enzyme, thymidylate synthase (TS), from E. coli. TS methylates 22-deoxyuridine 52-monophosphate (dUMP) to yield 22- deoxythymidine 52-monophosphate (dTMP), and it is highly conserved from bacteria to humans. Thymidine biosynthesis is a critical metabolic step that, if inhibited, results in cell death. Hence TS is targeted by anticancer drugs and is a viable target for antimicrobials. TS is an obligate homodimer that has a complex, multistep reaction mechanism and displays negative cooperativity. Complex mechanistic features are commonly found in enzymes involved in cell metabolism, yet how these molecules traverse complex energy landscapes to fulfill their function is not well understood. Currently, metabolic enzymes such as TS have not been subjected to thorough analysis of their internal conformational dynamics, largely due to their size. Characterization of TS therefore represents an early example of detailed study of the functional motions of a large metabolic enzyme. The mechanism of TS can be broken down into ~7 distinct steps. Intermediates can be trapped via specific combinations of substrate modifications and mutations, and these intermediates will be studied for their dynamic properties along the reaction coordinate. A key component of the approach will be to not only monitor protein, but to also monitor motions of substrates appropriately labeled for NMR relaxation. Using small molecules as probes will allow active-site motion of all trapped intermediates to be examined in an efficient manner. In aim 1, the effect of substrate binding and bond making/breaking on ¿s-ms and ps-ns motions will be tracked throughout TS using backbone and methyl-based NMR relaxation. In aim 2, the basis for negative cooperativity in TS will be examined from dynamical and steady-state kinetics perspectives. Key to this approach is the generation of mixed asymmetric dimers. In aim 3, previous findings on dihydrofolate reductase (DHFR) will be extended to probe the specific mechanism (and structure) of inhibitor dissociation. A novel relaxation dispersion approach will be taken that will enhance sensitivity to motions that mediate ligand dissociation. Our understanding of enzyme function is growing based on investigation of dynamics in small enzymes. An analogous approach is therefore needed to examine the role of dynamics in larger, more complex enzymes, which should lead to new strategies in drug design and protein engineering.