REACTIVE INTERMEDIATES OF ENZYMATIC REACTIONS

酶反应的反应中间体

• 批准号: 7788817
• 负责人: John P Richard
• 金额: $ 36.01万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7788817
• 关键词: 3-hydroxybutanal; 5 fluorouridine; Acceleration; Active Sites; Address; Affinity; Amino Acids; Binding; Binding Sites; Biochemical Reaction; Buffaloes; Carbon; Carboxy-Lyases; Catalysis; Complement; Complex; Decarboxylation; Disease; Distant; Drug Design; Eating; Elements; Enzyme Activation; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Goals; Homologous Gene; Isomerase; Ketoses; Ketosis; Kinetics; Knowledge; Length; Ligands; Maps; Metabolic Pathway; Metric; Modeling; Molecular; Motion; Movement; Mutation; Organism; Phosphites; Physical condensation; Protein Dynamics; Proteins; Protocols documentation; Protons; Pyrimidine; Pyrimidines; Reaction; Relative (related person); Resolution; Role; Side; Site; Site-Directed Mutagenesis; Specificity; Triose-Phosphate Isomerase; Trioses; base; carbanion; carboxylate; catalyst; chemical reaction; deprotonation; design; enzyme substrate; epimerase; epimerization; flexibility; inorganic phosphate; interest; member; novel; orotidylic acid; public health relevance; research study; ribulose 5-phosphate; sugar; transmission process

DESCRIPTION (provided by applicant): The hallmark of enzyme catalysis is the high affinity of enzymes for their transition states, but the origin of this tight binding is generally not well understood. This application describes experiments to probe the mechanism by which the large intrinsic binding energy of substrate phosphodianion groups is utilized by a variety of enzymes to stabilize the transition state for formation of an unstable carbanion intermediate. Our central hypothesis is that flexible "phosphate gripper" loops are wide-spread conserved protein elements that provide binding energy that may be utilized for stabilization of the transition state for formation of an enzyme-bound carbanion. The enzymatic reactions of interest include aldose-ketose isomerization, sugar epimerization, aldol condensation and decarboxylation. We will use our "two-part substrate" protocol, where the substrate phosphodianion group, modeled by exogenous phosphite dianion, is detached from the portion of the substrate that undergoes chemical reaction. The specific transition state stabilization from phosphodianion binding interactions can then be quantified from the observed activation of the enzyme by exogenous phosphite dianion towards catalysis of the reaction of the second substrate fragment. Four projects are described to examine both the generality and the mechanism of transition state stabilization arising from flexible loop-phosphite interactions. (1) We will examine members of the orotidine 5'-monophosphate decarboxylase (OMPDC) superfamily. The goal is to determine whether the structurally conserved phosphate gripper loops of these enzymes share the common function of providing specific stabilization of carbanion intermediates. (2) We will examine the relationship between the length of the phosphate gripper loop and the utilization of enzyme-phosphodianion binding interactions for two enzymes that catalyze epimerization of phosphorylated sugars differing in length by only one carbon atom. (3) We will probe the mechanism by which OMPDC achieves its enormous 1017-fold rate acceleration for the chemically difficult decarboxylation of orotidine 5'-monophosphate. We will probe the mechanism by which interactions between the enzyme and the substrate phosphodianion are utilized in stabilization of the transition state for decarboxylation at the distant pyrimidine ring, and address other questions about the enigmatic mechanism of action of this enzyme. (4) We will continue our studies of the role of flexible loop-phosphodianion interactions in stabilization of the transition state for formation of the enediol(ate) intermediate of the aldose-ketose isomerization of triose phosphates catalyzed by triose phosphate isomerase. A major goal is to provide a full description of the physical mechanism by which the movement of flexible catalytic loops acts as a "switch" to turn on stabilizing transition state interactions. PUBLIC HEALTH RELEVANCE: Enzyme catalysts are one of the principal components of all living systems, and there are many diseases that arise from the malfunction or deficiency of only a single enzyme. Advances in the understanding of enzyme catalysis from mechanistic studies of enzymes and of nonenzymatic reactions may prove critical for drug design, to the understanding of metabolic pathways and diseases, and to the resolution of other health-related issues. The focus of this application is the critical role of flexible phosphate gripper loops in enzymatic catalysis and the results may spur efforts to develop novel enzyme inhibitors that specifically target these loops.

描述（由申请人提供）：酶催化的标志是酶对其过渡态的高亲和力，但这种紧密结合的起源通常不太清楚。本申请描述了探测底物磷二阴离子基团的大固有结合能被各种酶利用以稳定过渡态以形成不稳定碳阴离子中间体的机制的实验。我们的中心假设是，灵活的“磷酸夹”环是广泛分布的保守的蛋白质元素，提供结合能，可用于稳定的过渡态形成的酶结合碳负离子。感兴趣的酶促反应包括醛糖-酮糖异构化、糖差向异构化、羟醛缩合和脱羧。我们将使用我们的“两部分底物”协议，其中底物磷酸根阴离子基团，由外源性亚磷酸根二价阴离子建模，从经历化学反应的底物部分分离。然后，可以从观察到的外源性亚磷酸根二价阴离子对第二底物片段的反应的催化对酶的活化来量化来自磷酸二价阴离子结合相互作用的特定过渡态稳定。四个项目被描述为检查的一般性和灵活的环亚磷酸酯相互作用所产生的过渡态稳定的机制。(1)我们将研究乳清酸核苷5 '-单磷酸脱羧酶（OMPDC）超家族的成员。我们的目标是确定这些酶的结构保守的磷酸夹环是否具有提供碳负离子中间体的特定稳定化的共同功能。(2)我们将研究磷酸夹环的长度和利用酶磷酸二阴离子结合相互作用的两种酶，催化差向异构化的磷酸化糖的长度不同，只有一个碳原子之间的关系。(3)我们将探索OMPDC实现其巨大的1017倍速率加速的化学困难的乳清酸核苷5 '-单磷酸脱羧的机制。我们将探讨的机制，酶和底物之间的相互作用，利用在稳定的过渡态脱羧在遥远的嘧啶环，并解决其他问题的神秘机制，这种酶的行动。(4)我们将继续研究磷酸丙糖异构酶催化的磷酸丙糖的醛酮异构化反应中，柔性环-磷酸双阴离子相互作用在形成包被（酯）中间体的过渡态稳定中的作用。一个主要的目标是提供一个完整的描述的物理机制，灵活的催化环的运动作为一个“开关”，打开稳定的过渡态相互作用。公共卫生相关性：酶催化剂是所有生命系统的主要组成部分之一，有许多疾病是由于单一酶的故障或缺乏而引起的。从酶和非酶反应的机理研究中了解酶催化的进展可能对药物设计，对代谢途径和疾病的理解以及对其他健康相关问题的解决至关重要。该应用的重点是柔性磷酸盐夹环在酶催化中的关键作用，其结果可能会刺激开发专门针对这些环的新型酶抑制剂的努力。