MECHANISMS FOR ENZYME CATALYSIS OF HETEROLYTIC REACTION

提出了几个与该机制直接相关的问题 对于醛糖-酮糖异构化反应的酶催化， 糖基转移反应和羟醛缩合反应。 的 这些问题的解决方案已经回避了采用一些 机械酶学最复杂的实验方法。 本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)目前尚不清楚是否非酶或酶催化 通过氢化物或质子进行糖的异构化反应 转移机制，或者如果一般的酸和金属离子是有效的 这些反应的催化剂 关于非酶反应的问题 将通过研究甘油醛的异构化和 相关基板 质子和氢化物转移反应赢得了 通过使用氘或氚标记的化合物来区分。 一般的酸和金属离子的催化作用也将进行研究。(2)是 尚不清楚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 对于每一个键合的变化，或者通过一个协调的机制， 在单个步骤中发生更多的键合变化。 这些区别是 必要的定义的作用的催化必要的 氨基酸残基Glu-461和Tyr-503。 循序渐进 亲核试剂加到半乳糖基酶上的机理 中间体（E-Gal）将在以下反应的研究中区分： 亲核阴离子 逐步建立协调一致的机制， 碱催化的酰基醛键的裂解将通过以下特征来区分： 改变亲核试剂碱性的效果的测定 对E-Gal对烷基醇和酚的反应性的影响，以及 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在 二维反应坐标轮廓。(3)的障碍， 烯醇化物离子与羰基在水中的加成反应将 确定，以评估它是否有利于酶 催化剂（例如，醛缩酶）来稳定这种过渡态 反应 缓冲酸作为催化剂的有效性 也将确定羰基的烯醇化物。 在许多场合已经证明，酶的研究 机制可以为药物设计提供关键信息（酶 抑制剂），了解代谢疾病，并 解决其他健康问题。

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.