Studies on Enzyme Activation and Novel Modes of Inhibition

近年来，酶机制的研究进展缓慢，部分原因是研究人员失败了 明确界定为达成最终结论而必须解决的所有重要问题 关于这些反应机制。这项拨款中描述的许多研究都是为了利用 旨在回答以下问题的创造性工作的潜力：“酶是如何实现其 过渡态(TS)结合的特异性？“这一潜力已经在布法罗的研究中得到了利用 磷酸丙糖异构酶和5‘-单磷酸脱羧酶的作用机制 和3-磷酸甘油脱氢酶(GPDH)。这些酶经历了双阴离子驱动的构象 从松散的，无连接的，开放的酶到僵硬的，有结构的，催化活性的闭合形式，它们起作用 作为打开紧密过渡状态结合相互作用的表达的开关。四个关键问题是 本文的目标是将TIM、OMPDC和GPDH的早期结论推广到其他 酶，并启动新的研究，以开发新的抑制剂TIM和OMPDC从致病菌 有机体。关键问题1：还有哪些蛋白质催化剂利用其非反应底物的结合作用 驱动酶激活构象变化的片段？这些实验将探索 来自底物腺苷环的结合能，或来自 酒精脱氢酶的底物，在这些酶催化过程中驱动构象变化， 充当打开紧密过渡状态绑定交互的开关。关键问题2： TIM、OMPDC和GPDH的催化和活化位置使其能够利用固有的底物结合 用于催化的能量？描述了一种涉及到的氨基酸侧链网络的实验。 GPDH催化，结合活化OMPDC的机理 酶与底物5‘-一磷酸鸟苷核糖羟基的相互作用 (OMP)和5-F-OMP。关键问题3：是否有足够先进的计算方法来模拟 TIM和GPDH催化的反应激活屏障上的定点突变？计算将是 与瑞典乌普萨拉的Lynn Kamerlin教授合作进行的，以确定现有的 EVB方法能够模拟这些酶的广泛突变研究的结果，目的是 扩大这些计算方法的限制。关键问题4：选择的潜力有多大 对病原生物TIM和OMPDC的抑制具有物种特异性的多肽？ 与日本东京大学的菅义明教授合作，提议进行实验，以 鉴定布鲁氏锥虫TIM和恶性疟原虫OMPDC的物种特异性抑制物， 并用X射线结晶学和计算方法表征了重要的抑制剂与蛋白质的相互作用 对接研究。

Progress in studies of enzyme mechanisms has slowed in recent years, in part because investigators have failed to clearly define all of the important questions that must be addressed in order to move towards final conclusions about these reaction mechanisms. Many of the studies described in this grant are designed to leverage the potential for creative work directed towards answering the following question: "How do enzymes achieve their specificity in transition state (TS) binding?" This potential has been harnessed in studies in Buffalo on the mechanism of action of triosephosphate isomerase (TIM), orotidine 5'-monophosphate decarboxylase (OMPDC) and glycerol 3-phosphate dehydrogenase (GPDH). These enzymes undergo dianion-driven conformational changes from loose, unliganded, open enzymes to stiff, structured, catalytically active closed forms, which act as “switches” that turn on the expression of tight transition state binding interactions. Four key question are addressed in this grant, with the goals of generalizing earlier conclusions from TIM, OMPDC, and GPDH to other enzymes, and of initiating new studies to develop novel inhibitors of TIM and OMPDC from pathogenic organisms. Key Question 1: What other protein catalysts utilize binding interactions of their nonreacting substrate fragments to drive enzyme-activating conformational changes? These experiments will probe whether the binding energy from the adenosine ring of the substrate for adenylate kinase, or from the NAD cofactor of the substrate for alcohol dehydrogenase, which drive conformational changes during catalysis by these enzymes, act as a switch to turn on tight transition state binding interactions. Key Question 2: What interactions between the catalytic and activation sites of TIM, OMPDC and GPDH enable utilization of the intrinsic substrate binding energy for catalysis? Experiments are described to characterize a network of amino acid side chains involved in catalysis by GPDH, and to characterize the mechanism for activation of OMPDC by the utilization of binding interactions between the enzyme and the ribosyl hydroxyl groups of the substrates orotidine 5'-monophosphate (OMP) and 5-F-OMP. Key Question 3: Are computational methods sufficiently advanced to model the effect of site-directed mutations on the activation barrier for reactions catalyzed by TIM and GPDH? Calculations will be carried out in collaboration with Professor Lynn Kamerlin in Uppsala, Sweden, to determine whether existing EVB methods are able to model the results of extensive mutagenesis studies on these enzymes, with the goal of expanding the limits of these computational methods. Key Question 4: What is the potential for selection of peptides that show species specificity for inhibition of TIM and OMPDC from pathogenic organisms? Experiments are proposed, in collaboration with Professor Hiroaki Suga at the University of Tokyo, Japan, to identify species-specific inhibitors for TIM from Trypanosoma brucei and OMPDC from Plasmodium falciparum, and to characterize the important inhibitor-protein interactions by X-ray crystallography and computational docking studies.