MECHANISMS OF ENZYMES AND NONENZYMIC THIAMIN REACTIONS

建议对酶催化和非酶作用的机理进行探讨。 辅酶硫胺素(维生素B1)的反应。的长期目标是 这项拟议的研究是为了确定原子的内禀结合能如何 底物(S)和辅酶依赖硫胺素激活催化 酶；特别感兴趣的是内在结合能的作用 稳定或避免不稳定的碳负离子“中间体”，如 C(2)-叶立德和C(α)-碳负离子/烯胺。眼前的、短期的 目标是了解催化的激活是如何通过 硫胺素依赖酶上辅酶的不同部分 涉及这些碳负离子“中间体”的反应，并确定 是否来自中性三环形式的 辅酶(TN)应包括在影响因素的核算中 以激活硫胺素依赖的酶催化。 今后的研究将集中在酶促羟醛缩醛的作用机理和催化作用上。 涉及C(2)-叶立德和C(α)-的类型加成-消除反应 碳负离子/烯胺，以及这些作用机制的程度 加成-消除反应由非共价相互作用决定 影响细胞寿命的酶和辅酶之间的关系 碳负离子“中间体”。使用标准的动力学方法，同位素探针， 和交替辅酶(3，4-dimethyl-5-(2-pyrophosphoethyl)thiazolium Ion，DMT)在存在和不存在类似物的情况下 硫胺素的氨基嘧啶(AP)(4-氨基-2-甲基嘧啶，AMP)， (I)去除两个官能团之间的共价键的效果 硫胺素的结构域和(Ii)在自由基上去除硫胺素的AP部分 羟醛类加成-消除反应的能量-反应坐标图 丙酮酸脱羧酶催化的反应将被确定。这 信息将被用来估计PDC结合的DMT中C(2)-H的pKa， 确定C(2)-叶立德和C(α)碳负离子/烯胺是否具有 在存在和存在的情况下使用DMT重建的PDC上的显著寿命 AMP的缺失，并评估AP部分结合的贡献 激活酶的催化作用。第二，《公约》的约束机制 硫胺素到大肠杆菌周质硫胺素结合蛋白将被检测为 一种特别简单的硫胺素依赖酶模型 动力学和核磁共振方法以及同位素探针来确定结合 TN的价值是重要的。

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 the intrinsic binding energy of the substrate(s) and coenzyme activates catalysis by thiamin-dependent enzymes; a particular interest is the role of the intrinsic binding energy in stabilizing or avoiding such unstable carbanion "intermediates" as the C(2)-ylide and the C(alpha)-carbanion/enamine. The immediate, short-term goals are to understand how activation of catalysis is brought about by different portions of the coenzyme on a thiamin-dependent enzyme for reactions involving these carbanion "intermediates" and to determine whether contributions from binding of a neutral tricyclic form of the coenzyme (TN) should be included in the accounting of factors contributing to activation of thiamin-dependent enzyme catalysis. The research will focus on the mechanism and catalysis of enzymic aldol- type addition-elimination reactions involving the C(2)-ylide and C(alpha)- carbanion/enamine, and the extent to which the mechanisms of these addition-elimination reactions are determined by noncovalent interactions between the enzyme and coenzyme that influence the lifetime of the carbanion "intermediate". Using standard kinetic methods, isotopic probes, and an alternate coenzyme (3,4-dimethyl-5-(2-pyrophosphoethyl)thiazolium ion, DMT) in the presence and absence of an analogue of the aminopyrimidinyl moiety (AP) of thiamin (4-amino-2-methylpyrimidine, AMP), the effect of (i) removing the covalent link between the two functional domains of thiamin and (ii) removing the AP moiety of thiamin on the free energy-reaction coordinate diagrams for aldol-type addition-elimination reactions catalyzed by pyruvate decarboxylase will be determined. This information will be used to estimate the pKa of C(2)-H in PDC-bound DMT, determine whether the C(2)-ylide and C(alpha)carbanion/enamine have a significant lifetime on PDC reconstituted with DMT in the presence and absence of AMP, and evaluate the contribution of binding of the AP moiety to activation of enzyme catalysis. Second, the mechanism for binding of thiamin to E. coli periplasmic thiamin-binding protein will be examined as an especially simple model for thiamin-dependent enzymes using standard kinetic and NMR methods and isotopic probes to determine whether binding of TN is important.