REACTIVE INTERMEDIATES OF ENZYMATIC REACTIONS

酶反应的反应中间体

基本信息

  • 批准号:
    8068750
  • 负责人:
  • 金额:
    $ 35.65万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 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

项目摘要

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(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还不是很清楚。本申请描述了探索底物磷二阴离子基团的大固有结合能被各种酶利用来稳定形成不稳定碳负离子中间体的过渡态的机制的实验。我们的中心假设是,灵活的“磷酸夹持器”环是广泛分布的保守蛋白质元件,提供结合能量,可用于稳定过渡态,形成酶结合的碳负离子。酶促反应包括醛糖-酮糖异构化、糖异构化、羟醛缩合和脱羧化。我们将使用我们的“两部分底物”方案,其中底物磷二阴离子基团,由外源亚磷酸二阴离子模拟,从经历化学反应的底物部分分离。然后,从观察到的外源性亚磷酸二阴离子对酶的激活到第二底物片段反应的催化作用,可以定量地描述从磷二阴离子结合作用中的特定过渡态稳定性。描述了四个项目,以研究由柔性环-亚磷酸盐相互作用引起的过渡态稳定的一般性和机制。(1)我们将研究5‘-一磷酸核苷脱羧酶超家族的成员。目的是确定这些酶的结构保守的磷酸盐夹持环是否具有提供碳负离子中间体特定稳定的共同功能。(2)我们将研究两种酶的磷酸钳环长度与酶-磷二阴离子结合作用的利用之间的关系,这两种酶催化长度仅相差一个碳原子的磷酸化糖的异构化。(3)我们将探讨OMPDC对化学上困难的5‘-单磷酸核苷的脱羧化反应达到1017倍的巨大提速的机制。我们将探索酶与底物磷阴离子之间的相互作用在稳定远距离嘧啶环上脱羧基的过渡态中的作用机制,并解决有关该酶作用机制的其他谜团问题。(4)我们将继续研究柔性环-磷二阴离子相互作用在磷酸丙糖异构酶催化磷酸三糖醛-酮糖异构化过渡态生成烯二醇(ATE)中间体中的作用。一个主要的目标是提供一个完整的物理机制的描述,通过这个机制,柔性催化环的移动起到“开关”的作用,开启稳定的过渡态相互作用。与公众健康相关: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。从酶的机理研究和非酶反应的机理研究中了解酶催化的进展可能被证明对药物设计、新陈代谢途径和疾病的理解以及其他与健康有关的问题的解决至关重要。这一应用的重点是柔性磷酸盐夹持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。

项目成果

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John P Richard其他文献

John P Richard的其他文献

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{{ truncateString('John P Richard', 18)}}的其他基金

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
  • 财政年份:
    2020
  • 资助金额:
    $ 35.65万
  • 项目类别:
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
  • 财政年份:
    2020
  • 资助金额:
    $ 35.65万
  • 项目类别:
Activation of Enzymes for Catalysis: The Role of Substrate-Induced Structural Changes
催化酶的激活:底物诱导的结构变化的作用
  • 批准号:
    9198549
  • 财政年份:
    2016
  • 资助金额:
    $ 35.65万
  • 项目类别:
Ribozymes for new genetic coding systems
用于新遗传编码系统的核酶
  • 批准号:
    7188027
  • 财政年份:
    2000
  • 资助金额:
    $ 35.65万
  • 项目类别:
Ribozymes for new genetic coding systems
用于新遗传编码系统的核酶
  • 批准号:
    7012204
  • 财政年份:
    2000
  • 资助金额:
    $ 35.65万
  • 项目类别:
Ribozymes for new genetic coding systems
用于新遗传编码系统的核酶
  • 批准号:
    6844940
  • 财政年份:
    2000
  • 资助金额:
    $ 35.65万
  • 项目类别:
MECHANISMS FOR ENZYME CATALYSIS OF HETEROLYTIC REACTION
酶催化杂解反应的机理
  • 批准号:
    2184725
  • 财政年份:
    1992
  • 资助金额:
    $ 35.65万
  • 项目类别:
MECHANISMS FOR ENZYME CATALYSIS OF HETEROLYTIC REACTION
酶催化杂解反应的机理
  • 批准号:
    2184726
  • 财政年份:
    1992
  • 资助金额:
    $ 35.65万
  • 项目类别:
MECHANISMS FOR ENZYME CATALYSIS OF HETEROLYTIC REACTIONS
酶催化杂解反应的机制
  • 批准号:
    3306773
  • 财政年份:
    1992
  • 资助金额:
    $ 35.65万
  • 项目类别:
MECHANISMS FOR ENZYME CATALYSIS OF HETEROLYTIC REACTIONS
酶催化杂解反应的机制
  • 批准号:
    3306774
  • 财政年份:
    1992
  • 资助金额:
    $ 35.65万
  • 项目类别:
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