Studies on Enzyme Activation and Novel Modes of Inhibition

酶激活和新抑制模式的研究

• 批准号: 10317064
• 负责人: John P Richard
• 金额: $ 39.54万
• 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/10317064
• 关键词: Acceleration; Address; Adenosine; Alcohol dehydrogenase; Amino Acids; Binding; Biochemical Reaction; Buffaloes; Carboxy-Lyases; Catalysis; Collaborations; Computing Methodologies; 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.

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 明确界定为得出最终结论而必须解决的所有重要问题 关于这些反应机制。这笔赠款中描述的许多研究旨在利用 创造性工作的潜力旨在回答以下问题：“酶如何实现其作用 过渡态（TS）结合的特异性？”这种潜力已在布法罗的研究中得到利用 磷酸三糖异构酶（TIM）、乳清苷5'-单磷酸脱羧酶（OMPDC）的作用机制 和甘油3-磷酸脱氢酶（GPDH）。这些酶经历双阴离子驱动的构象 从松散的、无配体的、开放的酶转变为僵硬的、结构化的、具有催化活性的封闭形式，其作用 作为打开紧密过渡态结合相互作用表达的“开关”。四个关键问题是 本次拨款中提出了解决方案，目标是将 TIM、OMPDC 和 GPDH 的早期结论推广到其他机构 酶，并启动新的研究，从病原体中开发 TIM 和 OMPDC 的新型抑制剂 有机体。关键问题 1：还有哪些其他蛋白质催化剂利用其非反应底物的结合相互作用 片段驱动酶激活构象变化？这些实验将探讨是否 来自腺苷酸激酶底物的腺苷环或来自腺苷酸激酶的 NAD 辅因子的结合能 乙醇脱氢酶的底物，在这些酶的催化过程中驱动构象变化， 充当打开紧密过渡态结合相互作用的开关。关键问题 2： 之间有哪些相互作用 TIM、OMPDC 和 GPDH 的催化和激活位点能够利用内在底物结合 催化能量？实验被描述为表征涉及氨基酸侧链的网络 GPDH 催化，并通过结合来表征 OMPDC 的激活机制 酶与底物乳清苷 5'-单磷酸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来模拟 TIM 和 GPDH 催化反应的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，以确定是否存在 EVB 方法能够对这些酶的广泛诱变研究结果进行建模，其目标是 扩大这些计算方法的局限性。关键问题 4：选择的潜力有多大？ 具有抑制病原生物体 TIM 和 OMPDC 的物种特异性的肽？ 与日本东京大学的 Hiroaki Suga 教授合作进行了实验，以 鉴定来自布氏锥虫的 TIM 和来自恶性疟原虫的 OMPDC 的物种特异性抑制剂， 并通过 X 射线晶体学和计算来表征重要的抑制剂-蛋白质相互作用 对接研究。