The Role of Dynamics in Enzyme Mechanism and Inhibition

描述（由申请人提供）：酶是驱动细胞过程的显著纳米机器。研究它们的基本性质是为了更好地理解催化作用，研究它们是为了提高我们设计针对它们的治疗方法的能力，以造福人类健康。经过几十年的结构生物学研究，我们对酶如何工作的理解仍在不断增长。最近的工作，特别是在NMR光谱学领域，例如，已经揭示了动态运动和预先存在的构象转换对于许多（如果不是全部）酶的功能至关重要。在这个建议中，最近开发的大蛋白质的NMR自旋弛豫研究方法，与小分子合成和稳态动力学配对，将应用于64 kD的酶，胸苷酸合成酶（TS），从E。杆菌TS甲基化22-脱氧尿苷52-单磷酸（dUMP）产生22-脱氧胸苷52-单磷酸（dTMP），并且其从细菌到人高度保守。胸苷生物合成是一个关键的代谢步骤，如果受到抑制，会导致细胞死亡。因此，TS被抗癌药物靶向，并且是抗微生物剂的可行靶标。TS是一种专性同二聚体，具有复杂的多步反应机制，并表现出负协同性。复杂的机械特征通常存在于参与细胞代谢的酶中，但这些分子如何穿越复杂的能量景观以实现其功能还不清楚。目前，代谢酶，如TS还没有进行彻底的分析，其内部构象动力学，主要是由于它们的大小。因此，TS的表征代表了详细研究大型代谢酶的功能运动的早期例子。TS的机制可以分为~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被捕获，并且这些中间体将被研究其沿着反应坐标的动力学性质。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为NMR弛豫的底物的运动。使用小分子作为探针将允许以有效的方式检查所有捕获的中间体的活性位点运动。在目标1中，将使用主链和基于甲基的NMR弛豫在整个TS中跟踪底物结合和键形成/断裂对<$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研究TS中负协同性的基础。这种方法的关键是混合不对称二聚体的产生。在目标3中，先前关于二氢叶酸还原酶（DHFR）的发现将被扩展到探测抑制剂解离的特定机制（和结构）。将采用一种新的弛豫色散方法， 调节配体解离的运动。我们对酶功能的理解是基于对小酶动力学的研究而增长的。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，这将导致药物设计和蛋白质工程的新策略。

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.