REACTIVE INTERMEDIATES OF ENZYMATIC REACTIONS

在水中的模型研究提出了表征动力学 反应性和平衡稳定性的高度不稳定的三元 碳正离子、羰基正离子和硫醇酯烯醇化物 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 虽然这些物种一直被认为是 酶促反应中间体，直接证据表明， 缺乏形成，有许多未回答的问题 关于它们在溶液中和在 酶活性位点 本文将解决以下问题： 提议 1)叔丁基和环取代的取代反应 将研究枯基衍生物以：a.确定 作为反应中间体形成的碳阳离子的寿命; B. 确定协同双分子取代的重要性 在叔碳上的反应;和c.表征溶液 叔碳阳离子的反应性， 异戊烯焦磷酸异构酶。 2)实验将 以确定是否有一个简单的关系， 氧碳离子寿命，和一般酸的发生， 碱催化正碳离子的形成和分解。 这些模型研究将确定 一般的酸碱催化有助于酶催化， 糖基裂解。 3)生成的速率常数和 对于缓冲液，将测定硫醇酯烯醇化物的分解， 和溶剂催化的反应，并结合起来，得到pKa为 碳去质子化 烯醇化物的寿命， 将估计缓冲酸，以确定是否 这些碳负离子足够稳定，可以作为 酶催化克莱森缩合反应。 （14C）─ 标记的硫羟酸酯烯醇化物将在存在 酶如硫解酶及其共底物，以确定 如果烯醇化物在动力学上有能力作为反应 中间体 它已经被证明了无数次， 对酶机制的基础研究可能会发挥深刻的作用- 在解决健康相关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.