REACTIVE INTERMEDIATES OF ENZYMATIC REACTIONS

建议在水中进行模型研究来表征动力学。 高度不稳定的三元体系的反应性和平衡稳定性 碳正离子、氧碳离子和硫醇酯烯酸 酶催化的1，3烯丙基重排的中间体， 糖基转移和克莱森缩合反应， 分别进行了分析。尽管这些物种长期以来一直被认为 酶反应中间体，其直接证据 编队不足，还有许多悬而未决的问题。 关于它们在溶液中和在一个 酶活性部位。在本报告中解决了以下问题 求婚。1)叔丁基和环取代的取代反应 将对异丙基衍生物进行研究，以便：a.确定 作为反应中间体形成的碳正离子的寿命； 确定协同双分子替代的重要性 叔碳上的反应；以及c.表征溶液 叔碳正离子在研究中的反应性 异戊烯基焦磷酸异构酶。2)实验将是 执行以确定以下各项之间是否存在简单关系 氧碳离子寿命，以及一般酸的出现- 碱性催化作用下耳石正离子的形成和击穿。 这些模型研究将确定在什么情况下 一般酸碱催化作用对酶催化的贡献 糖基裂解。3)地层和地层的速率常数 对于缓冲液，将测定硫醇酯烯醇的分解。 和溶剂催化的反应，并结合在一起得到了 碳去质子化。在存在的情况下的冷淡的寿命 将对缓冲酸进行估计，以确定 这些碳负离子足够稳定，可以作为 酶催化的克莱森缩合反应。(14C)- 标记的硫醇酯将在存在的情况下生成 硫解酶等酶及其共底物的测定 如果烯醇基团在动力学上有能力作为反应 中级的。它已经被无数次地证明 酶机制的基础研究可能会发挥更深层次的作用- 在解决与健康相关的问题方面发挥重要作用。

Model studies in water are proposed to characterize the kinetic reactivity, and equilibrium stability of highly unstable tertiary carbocation, oxocarbonium ion and thiol ester enolate intermediates of enzyme-catalyzed 1,3 allylic rearrangements, glycosyl transfer, and Claisen condensation reactions, respectively. Although these species have long been putative enzymatic reaction intermediates, direct evidence for their formation is lacking, and there are many unanswered questions about the nature of their existence both in solution and at an enzyme active site. The following problems are addressed in this proposal. 1) Substitution reactions at t-butyl and ring-substituted cumyl derivatives will be studied in order to: a. Determine the lifetimes for carbocations that form as reaction intermediates; b. Determine the importance of concerted bimolecular substitution reactions at tertiary carbon; and c. Characterize the solution reactivity of tertiary carbocations in preparation for studies on isopentenyl pyrophosphate isomerase. 2) Experiments will be performed to determine if there is a simple relationship between oxocarbonium ion lifetime, and the occurrence of general acid- base catalysis of otocarbonium ion formation and breakdown. These model studies will define the conditions under which general acid-base catalysis contributes to enzymatic catalysis of glycosyl cleavage. 3) The rate constants for the formation and breakdown of a thiol ester enolate will be determined for buffer- and solvent-catalyzed reactions, and combined to give the pKa for carbon deprotonation. The enolate lifetime in the presence of buffer acids will be estimated in order to establish whether or not these carbanions are stable enough to exist as intermediates of enzyme catalyzed claisen condensation reactions. The (14C)- labelled thiol ester enolate will be generated in the presence of an enzyme such as thiolase and its cosubstrate in order to determine if the enolate is kinetically competent to serve as a reaction intermediate. It has been demonstrated numerous times the fundamental studies on enzyme mechanisms may play a deep- seated role in solving health related problems.