Studies on Enzyme Activation and Novel Modes of Inhibition

酶激活和新抑制模式的研究

• 批准号: 10543563
• 负责人: John P Richard
• 金额: $ 39.53万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10543563
• 关键词: Acceleration; Address; Adenosine; Alcohol dehydrogenase; Amino Acids; Binding; Biochemical Reaction; Carboxy-Lyases; Catalysis; Collaborations; Computing Methodologies; Creativeness; Development; Disease; Docking; Drug Design; Enzyme Activation; Enzymes; Glycerol-3-Phosphate Dehydrogenase; Goals; Grant; Health; Japan; Metabolic; Metabolic Diseases; Metabolic Pathway; Methods; Modeling; Molecular Conformation; Mutagenesis; Mutation; Organism; Pathogenicity; Peptides; Plasmodium falciparum; Proteins; Reaction; Research Personnel; Resolution; Side; Site; Species Specificity; Specificity; Structure; Sweden; Tokyo; Triose-Phosphate Isomerase; Trypanosoma brucei brucei; Universities; Work; X-Ray Crystallography; adenylate kinase; catalyst; cofactor; computer studies; design; enzyme mechanism; experimental study; hydroxyl group; inhibitor; novel; orotidylic acid; professor

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.

酶机制研究的进展近年来有所放缓，部分原因是研究人员失败了 要清楚定义所有必须解决的重要问题才能朝着最终结论迈进 关于这些反应机制。该赠款中描述的许多研究旨在利用 致力于回答以下问题的创造性工作的潜力：“酶如何实现其 过渡状态（TS）结合的特异性？”布法罗的研究中利用了这种潜力 三氧磷酸异构酶（TIM），甲磷酸5'-单磷酸脱羧酶（OMPDC）的作用机理 和3-磷酸甘油脱氢酶（GPDH）。这些酶经历了dianion驱动的构象 从松散，无配合，开放式酶变为僵硬，结构化的催化活性封闭形式，它们的作用 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 在这笔赠款中解决的目标是将蒂姆，opdc和gpdh的早期结论概括为其他 酶，以及启动新研究以从致病性开发TIM和OMPDC的新型抑制剂 有机体。关键问题1：其他哪些蛋白质催化剂利用其非反应底物的结合相互作用 碎片以驱动酶激活会议变化？这些实验将探测是否 来自腺苷酸激酶的腺苷环的结合能，或从NAD辅助因子的结合能 酒精脱氢酶的底物，这些酶在催化过程中驱动构象变化， 充当打开紧密过渡状态结合相互作用的开关。关键问题2：之间什么相互作用 TIM，OMPDC和GPDH的催化和激活位点可利用固有的底物结合 催化的能量？描述了实验以表征参与的氨基酸侧链网络 GPDH催化，并通过利用结合来表征OMPDC激活的机制 底物5'-单磷酸盐的酶和核糖基羟基之间的相互作用 （OMP）和5-f-op。关键问题3：计算方法是否适当提前以建模 TIM和GPDH催化反应的激活屏障上的位置突变？计算将是 与瑞典Uppsala的Lynn Kamerlin教授合作，以确定是否存在 EVB方法能够对这些酶进行广泛的诱变研究的结果进行建模，其目标 扩大这些计算方法的限制。关键问题4：选择的潜力是什么 表现出对抑制病原生物抑制TIM和OMPDC的规格特异性的肽？ 通过与日本东京大学的Hiroaki Suga教授合作提出实验 鉴定出来自Brucei锥虫的TIM的特异性抑制剂，来自恶性疟原虫的tim抑制剂， 并通过X射线晶体学和计算来表征重要的抑制剂 - 蛋白质相互作用 对接研究。