MECHANISMS OF ENZYMIC AND NONENZYMIC THIAMIN REACTIONS

建议对酶催化的反应机理进行研究。 以及辅酶硫胺素(维生素B1)的非酶反应。这个 拟议研究的长期目标是确定如何 依赖硫胺素的酶稳定或避免这种不稳定 碳负离子的“中间体”，如四氢叶立德和C。 α-碳负离子/烯胺与多起 酶机制；特别感兴趣的是，如果有的话，它的作用 底物(S)或辅酶的内禀结合能 稳定碳负离子。眼前的短期目标是 了解两种非酶的作用机理和催化作用 涉及这些中等不稳定的碳负离子的反应和 确定这些碳负离子是否以离散形式存在 依赖硫胺素的酶上的中间体。 今后的研究将集中在酶的作用机理和催化作用等方面。 和非酶的羟醛加成反应，涉及 四氢叶立德，以及在多大程度上 这些加成反应由 叶立德类碳负离子“中间体”。使用标准动能 方法和同位素探针，模型羟醛-型的机理 四氢叶立德与醛的加成反应 对水溶液进行测定，并与机理进行比较 硫胺素依赖的α-酮酸脱羧基 丙酮酸脱羧酶(PDC)。过渡期的结构 叶立德与醛的非酶加成状态为 表征，PDC结合的硫胺素C2-H的PA将为 估计。 第二，非酶Cα-质子转移的机理 四氢噻唑甲醇形成Cα-碳负离子/烯胺 水溶液将使用标准动力学方法进行检测。 和同位素探针来确定一般碱基的作用 催化、诱导效应和内部回报。这 信息将被用来估计Cα质子的pa， 确定Cα-碳负离子是否具有显著的寿命， 由此估算质子转移的本征势垒 亚胺离子-活性碳酸，并测定其效果 立体电子控制(轨道适当重叠的要求) 关于提取Cα的速率和内在障碍 水溶液中的质子。使用同位素探测器，它将是 已确定Cα-碳负离子是否具有显著的寿命 在PDC上。

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.