REACTIVE INTERMEDIATES OF ENZYMATIC REACTIONS

酶反应的反应中间体

• 批准号: 2180010
• 负责人: John P Richard
• 金额: $ 14.12万
• 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 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2180010
• 关键词: carbanion; carbonyl group; catalyst; chemical addition; chemical elimination; chemical kinetics; chemical reaction; enol; enolate; enzyme mechanism; enzyme model; glyoxal; isomerase; metals; protonation; quinones; sugar phosphates; thermodynamics; thioester; thiols; ultraviolet spectrometry

Model studies in water to characterize the kinetic reactivity, equilibrium stability and mechanisms for the formation and reaction of putative carbanion, carbocation and quinone methide intermediates of enzyme-catalyzed reactions are proposed. The following problems will be studied: (1) Oxygen and thiol ester enolates have been proposed as intermediates for a wide range of enzymatic reactions. However, there is scant direct evidence for their formation in water or at enzyme active sites. We will generate the putative enolate intermediates of seven enzymatic reactions by general base-catalyzed deprotonation of the respective carbonyl precursors, and determine the relative thermodynamic stabilities of these enolates. (2) The relative advantage for metal ion catalysis of deprotonation at carbon is not known. This will be determined by a comparison of metal-ion activation of proton transfer with the activation provided by a CH3 + group. (3) A series of alpha-substituted enol phosphotriesters will be synthesized and their potential use as precursors to alpha-substituted enols and ketenes, or as suicide enzyme inhibitors will be explored. (4) The enzymatic source of methylglyoxal in mammalian tissues will be purified and characterized in order to determine whether it is identical with triosephosphate isomerase. This work may resolve the enigma of the metabolic role of glyoxalase I and II. (5) It is not known if the reaction catalyzed by isopentenyl pyrophosphate isomerase proceeds through a tertiary carbocation reaction intermediate or if it proceeds through a highly polar, carbocation-like, transition state of a concerted reaction mechanism. We will determine whether a model nonenzymatic 1,3-allylic isomerization reaction follows a stepwise or a concerted mechanism. (6) The rate law for the reactions of a simple quinone methide with nucleophilic reagents will be characterized in order to determine if these reactions are subject to general acid and/or general base catalysis. The results of this work will be compared with results of studies on nucleophilic addition reactions at the carbonyl group. (7) Experiments will be performed to determine whether there is a simple relationship between carbocation lifetime and the occurrence of general acid-base catalysis of oxocarbenium ion formation and breakdown. Advances in the understanding of enzyme mechanisms that result from model studies of nonenzymatic reactions may prove critical for drug design (enzyme inhibitors), to the understanding of metabolic pathways and diseases, and to the resolution of other health-related problems.

水中的模型研究以表征动力学反应性， 平衡稳定性以及形成和反应的机制 假定的碳负离子、碳正离子和醌甲基化物中间体 提出了酶催化反应。 将会出现以下问题 研究：（1）氧和硫羟酸酯烯醇化物已被提议作为 用于多种酶促反应的中间体。 然而，有 缺乏直接证据证明它们在水中或在酶活性下形成 网站。 我们将生成七种假定的烯醇中间体 一般碱催化去质子化的酶促反应 各自的羰基前体，并确定相对热力学 这些烯醇化物的稳定性。 (2)金属离子的相对优势 碳去质子化的催化作用尚不清楚。 这将是 通过比较金属离子激活质子转移来确定 由CH3 + 基团提供激活。 (3)一系列 将合成α-取代烯醇磷酸三酯及其 潜在用途为α-取代烯醇和烯酮的前体，或作为 将探索自杀酶抑制剂。 (4)酶的来源 哺乳动物组织中的甲基乙二醛将被纯化并表征 以确定是否与磷酸三糖相同 异构酶。 这项工作可能会解开代谢作用之谜。 乙二醛酶 I 和 II。 (5) 尚不清楚该反应是否由 异戊烯焦磷酸异构酶通过三级异构酶进行 碳正离子反应中间体或如果它通过高度进行 协同反应的极性、类碳正离子、过渡态 机制。 我们将确定模型是否为非酶 1,3-烯丙基 异构化反应遵循逐步或协同机制。 (6) 简单醌甲基化物与 的反应速率定律 将表征亲核试剂以确定是否 这些反应受一般酸和/或一般碱的影响 催化。 这项工作的结果将与 羰基亲核加成反应的研究。 (7) 将进行实验以确定是否存在简单的 碳正离子寿命与一般现象发生的关系 氧碳鎓离子形成和分解的酸碱催化。 对模型产生的酶机制的理解取得进展 非酶反应的研究可能对药物设计至关重要 （酶抑制剂），以了解代谢途径和 疾病，并解决其他与健康相关的问题。