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未能 明确界定为达成最后结论而必须解决的所有重要问题， 这些反应机制。该补助金中描述的许多研究旨在利用 潜在的创造性工作，旨在回答以下问题：“酶如何实现其 过渡态结合的特异性？“这种潜力已经在布法罗的研究中得到了利用， 磷酸丙糖异构酶（TIM）、乳清酸核苷5 '-单磷酸脱羧酶（OMPDC）的作用机制 和甘油3-磷酸脱氢酶（GPDH）。这些酶经历二阴离子驱动的构象 从松散的、无配体的、开放的酶转变为刚性的、结构化的、催化活性的封闭形式， 作为“开关”，开启紧密过渡态结合相互作用的表达。四个关键问题是 在这项补助金中解决，与推广从TIM，OMPDC和GPDH到其他早期结论的目标 酶，并启动新的研究，以开发新的抑制剂的TIM和OMPDC从致病性 有机体关键问题1：还有哪些蛋白质催化剂利用它们的非反应底物的结合相互作用 片段来驱动酶激活构象变化？这些实验将探索 来自腺苷酸激酶底物的腺苷环或腺苷酸激酶的NAD辅因子的结合能。 醇脱氢酶底物，其在这些酶催化期间驱动构象变化， 作为一个开关来打开紧密的过渡态结合相互作用。关键问题2： TIM、OMPDC和GPDH的催化和活化位点使得能够利用内在底物结合 能源催化？描述了实验以表征涉及以下的氨基酸侧链的网络： 通过GPDH的催化作用，并通过利用结合来表征OMPDC活化的机制。 酶与底物乳清酸核苷5 '-单磷酸的核糖基羟基之间的相互作用 (OMP)和5-F-OMP。关键问题3：计算方法是否足够先进，可以模拟 在TIM和GPDH催化的反应的活化屏障上的定点突变？计算将 与瑞典乌普萨拉的林恩卡梅林教授合作进行，以确定是否存在 EVB方法能够模拟这些酶的广泛诱变研究的结果，目标是 扩展这些计算方法的极限。关键问题4：选择的潜力是什么 对病原生物的TIM和OMPDC的抑制显示出种属特异性的肽？ 与日本东京大学的Hiroaki Suga教授合作， 鉴定来自布氏锥虫TIM和来自恶性疟原虫的OMPDC的物种特异性抑制剂， 并通过X射线晶体学和计算表征重要的通道蛋白质相互作用 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.