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非凡纳米机器。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。在对酶进行了几十年的结构生物学研究之后，我们对它们如何工作的理解仍在增长。最近的工作，特别是在核磁共振光谱领域，例如，揭示了动态运动和预先存在的构象转换对许多酶的功能至关重要，如果不是全部的话。在本研究中，最近开发的大蛋白核磁共振自旋弛缓研究方法，结合小分子合成和稳态动力学，将应用于大肠杆菌64 kD酶胸苷酯合成酶（TS）。TS甲基化22-脱氧尿嘧啶52-单磷酸（dUMP）生成22-脱氧胸腺嘧啶52-单磷酸（dTMP），对细菌和人类具有高度保守性。胸腺嘧啶的生物合成是一个关键的代谢步骤，如果受到抑制，将导致细胞死亡。因此，TS是抗癌药物的靶点，也是抗微生物药物的可行靶点。TS是一种专性同二聚体，具有复杂的多步反应机制和负协同性。在参与细胞代谢的酶中通常发现复杂的机制特征，但这些分子如何穿越复杂的能量景观来实现其功能尚不清楚。目前，代谢酶如TS的内部构象动力学还没有得到深入的分析，很大程度上是由于它们的大小。因此，表征TS代表了对大型代谢酶的功能运动进行详细研究的早期例子。TS的机理可分为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，并且这些中间体将沿着反应坐标研究其动态性质。该方法的一个关键组成部分不仅是监测蛋白质，而且还监测适当标记为核磁共振弛豫的底物的运动。使用小分子作为探针可以有效地检测所有被捕获中间体的活性位点运动。在目标1中，底物结合和成键/断键对s-ms和ps-ns运动的影响将在整个TS过程中使用主链和甲基基核磁共振弛豫来跟踪。在目标2中，将从动力学和稳态动力学的角度研究TS负合作的基础。这种方法的关键是生成混合不对称二聚体。在目标3中，将扩展先前对二氢叶酸还原酶（DHFR）的研究结果，以探索抑制剂解离的具体机制（和结构）。本文将采用一种新的松弛色散方法来提高对

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.