MECHANISMS OF ENZYMIC AND NONENZYMIC THIAMIN REACTIONS

本文拟探讨酶促反应的机理 和辅酶硫胺素（维生素B1）的非酶反应。 的 拟议研究的长期目标是确定如何 硫胺素依赖性酶稳定或避免这种不稳定的 碳负离子“中间体”，如噻唑鎓 Cs-叶立德和C α-碳负离子/烯胺，其已经涉及许多 酶机制;特别感兴趣的是作用，如果有的话， 底物或辅酶的固有结合能， 稳定碳负离子。 近期目标是 了解两种非酶催化的机制和催化作用， 涉及这些中等不稳定的碳负离子的反应， 确定这些碳负离子是否以离散形式存在 一种依赖于硫胺素的酶的中间体。 本论文的研究重点是酶促反应的机理和催化作用 和非酶的羟醛型加成反应， 噻唑钙叶立德，以及在何种程度上的机制， 这些加成反应是由 叶立德型碳负离子“中间体”。 使用标准动力学 方法和同位素探针，一个模型的机理， 噻唑叶立德与醛的加成反应 水溶液将被确定，并与机理进行比较 α-酮酸的硫胺素依赖性脱羧作用， 丙酮酸脱羧酶（PDC）。 过渡的结构 叶立德与醛的非酶加成反应的状态将是 特征，并且PDC结合的硫胺素C2-H的pa将被 估算 第二，非酶C α-质子转移的机制， 噻唑鎓甲醇形成C α-碳负离子/烯胺， 将使用标准动力学方法检查水溶液 和同位素探针来确定总碱的作用 催化、诱导效应和内部回归。 这 信息将用于估计C α质子的Pa， 确定C α-碳负离子是否具有显著的寿命， 估计质子转移的固有势垒， 亚胺离子活性炭酸，并确定效果 立体电子控制（轨道适当重叠的要求） 对C α提取的速率和内在屏障的影响， 水溶液中的质子。 使用同位素探针， 确定C α-碳负离子是否具有显著的寿命 在PDC。

It is proposed to examine the mechanism of enzyme-catalyzed and nonenzymic reactions of the coenzyme thiamin (vitamin B1). The long-term objective of the proposed research is to determine how thiamin-dependent enzymes stabilize or avoid such unstable carbanion "intermediates" as the thiazolium Cs-ylide and the C alpha-carbanion/enamine that have been implicated in a number of enzyme mechanisms; a particular interest is the role, if any, of the intrinsic binding energy of the substrate(s) or coenzyme in stabilizing the carbanions. The immediate, short-term goals are to understand the mechanism and catalysis of two nonenzymic reactions involving these moderately unstable carbanions and to determine whether or not these carbanions exist as discrete intermediates on a thiamin-dependent enzyme. The research will focus on the mechanism and catalysis of enzymic and nonenzymic aldol-type addition reactions involving the thiazolium Ca-ylide, and the extent to which the mechanisms of these addition reactions are determined by the lifetime of the ylide-type carbanion "intermediate". Using standard kinetic methods and isotopic probes, the mechanism of a model aldol-type addition reaction between thiazolium ylides and aldehydes in aqueous solution will be determined and compared with the mechanism of thiamin-dependent decarboxylation of alpha-keto acids by pyruvate decarboxylase (PDC). The structure of the transition state for nonenzymic addition of the ylide to aldehydes will be characterized, and the pa of PDC-bound thiamin C2-H will be estimated. Secondly, the mechanism for nonenzymic C alpha-proton transfer from thiazolium carbinols to form the C alpha-carbanion/enamine in aqueous solution will be examined using standard kinetic methods and isotopic probes to determine the role of general base catalysis, inductive effects, and internal return. This information will be used to estimate the pa of the C alpha-proton, determine whether the C alpha-carbanion has a significant lifetime, estimate the intrinsic barrier for proton transfer from these iminium ion-activated carbon acids, and determine the effect of stereoelectronic control (a requirement for proper orbital overlap) on the rate and intrinsic barrier for abstraction of the C alpha- proton in aqueous solution. Using isotopic probes, it will be determined whether the C alpha-carbanion has a significant lifetime on PDC.