MECHANISMS FOR ENZYME CATALYSIS OF HETEROLYTIC REACTION

酶催化杂解反应的机理

• 批准号: 2184725
• 负责人: John P Richard
• 金额: $ 17.1万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2184725
• 关键词: aldehyde lyase; beta galactosidase; catalyst; chemical addition; chemical structure function; chemical transfer reaction; deuterium; enzyme mechanism; enzyme model; hydrogen; isomerase; tritium

Several problems are presented that are directly related to the mechanism for enzymatic catalysis of aldose-ketose isomerization reactions, glycosyl transfer reactions and aldol condensation reactions. The solutions to these problems have eluded investigations employing some of the most sophisticated experimental methods of mechanistic enzymology. This proposal describes how these problems may be resolved by an application of the tools and principles of physical organic chemistry. Three different projects are described. (1) It is not known whether the nonenzymatic or enzyme-catalyzed isomerization reactions of sugars proceed by a hydride or a proton transfer mechanism, or if general acids and metal ions are effective catalysts of these reactions. Questions about the nonenzymatic reactions will be resolved by a study of the isomerization of glyceraldehyde and a related substrate. Proton and hydride transfer reactions win be distinguished by use of deuterium or tritium-labelled compounds. Catalysis by general acids and metal ions will also be studied. (2) It is not known whether the glycosyl transfer reactions catalyzed by beta-galactosidase proceed by a mechanism with discrete chemical steps for every change in bonding, or by a concerted mechanism where two or more changes in bonding occur in a single step. These distinctions are necessary for the definition of the roles of the catalytically essential amino acid residues Glu-461 and Tyr-503. Stepwise and concerted mechanisms for the addition of nucleophiles to the galactosyl-enzyme intermediate (E-Gal) will be distinguished in a study of the reactions of nucleophilic anions. Stepwise and concerted mechanisms for general base-catalyzed cleavage of the acylal linkage will be distinguished by a determination of the effect of changing the basicity of the nucleophile on the reactivity of E-Gal toward alkyl alcohols and phenols, and the solvent deuterium isotope effects on these reactions. The transition state for the general base-catalyzed reaction will be characterized on a two-dimensional reaction coordinate profile. (3) The barrier for the addition reaction of an enolate ion to a carbonyl group in water will be determined, in order to assess whether it is advantageous for enzyme catalysts (e.g., aldolases) to stabilize the transition state for this reaction. The effectiveness of buffer acids as catalysts of addition of enolates to the carbonyl group will also be determined. It has been demonstrated on numerous occasions that studies on enzyme mechanisms may provide information critical for drug design (enzyme inhibitors), to the understanding metabolic diseases, and to the resolution of other health-related problems.

提出了与机制直接相关的几个问题 用于醛糖-酮糖异构化反应的酶催化， 糖基转移反应和羟醛缩合反应。 这 这些问题的解决方案未能通过某些研究进行调查 最复杂的机械酶学实验方法。 该提案描述了如何通过 物理有机化学的工具和原理的应用。 描述了三个不同的项目。 (1) 尚不清楚是非酶催化的还是酶催化的 糖的异构化反应通过氢化物或质子进行 转移机制，或者一般酸和金属离子是否有效 这些反应的催化剂。 关于非酶反应的问题 将通过甘油醛异构化的研究来解决 相关底物。 质子和氢化物转移反应获胜 通过使用氘或氚标记的化合物来区分。 还将研究一般酸和金属离子的催化作用。 (2) 是 不知道糖基转移反应是否由 β-半乳糖苷酶通过具有离散化学步骤的机制进行 对于联系的每一次变化，或者通过协调机制，其中两个或 在一个步骤中就会发生更多的粘合变化。 这些区别是 对于定义催化必需的作用是必要的 氨基酸残基Glu-461和Tyr-503。 循序渐进、协调一致 将亲核试剂添加到半乳糖基酶的机制 中间体（E-Gal）将在反应研究中被区分 亲核阴离子。 逐步协调的一般机制 酰基键的碱催化裂解可通过以下特征来区分： 测定改变亲核试剂碱度的影响 E-Gal 对烷基醇和酚的反应活性 溶剂氘同位素对这些反应的影响。 过渡 一般碱催化反应的状态将被表征为 二维反应坐标剖面。 (3) 障碍 烯醇离子与水中羰基的加成反应为 确定，以评估是否对酶有利 催化剂（例如醛缩酶）来稳定过渡态 反应。 缓冲酸作为加成催化剂的有效性 羰基的烯醇化物也将被测定。 酶的研究已被多次证明 机制可以提供对药物设计至关重要的信息（酶 抑制剂），了解代谢疾病，并 解决其他与健康相关的问题。