MECHANISMS OF ENZYMES AND NONENZYMIC THIAMIN REACTIONS

酶和非酶硫胺素反应的机制

• 批准号: 2181706
• 负责人: MICHAEL W WASHABAUGH
• 金额: $ 25.05万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2181706
• 关键词: acetaldehyde; aminopyrimidine; binding proteins; carbanion; carbon; chemical addition; chemical binding; chemical elimination; chemical kinetics; chemical stability; chemical structure function; cofactor; enzyme activity; enzyme mechanism; intermolecular interaction; ionic strengths; nonradiation isotope effect; nuclear magnetic resonance spectroscopy; protonation; pyruvate decarboxylase; pyruvates; thermodynamics; thiamine; thiamine pyrophosphate; ylide

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.

建议研究酶催化和非酶催化的机制 辅酶硫胺素（维生素 B1）的反应。长期目标 拟议的研究是确定内在结合能如何 底物和辅酶通过硫胺素依赖性激活催化 酶；特别感兴趣的是内在结合能的作用 稳定或避免此类不稳定的碳负离子“中间体” C(2)-叶立德和C(α)-碳负离子/烯胺。即时的、短期的 目标是了解催化的激活是如何引起的 硫胺素依赖性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 中性三环形式的结合是否有贡献 辅酶（TN）应包含在影响因素的计算中 激活硫胺素依赖性酶催化作用。 该研究将重点关注酶促醛醇缩合的机理和催化。 涉及 C(2)-叶立德和 C(α)- 的加成-消除反应 碳负离子/烯胺，以及这些机制的程度 加成-消除反应由非共价相互作用决定 影响酶寿命的酶和辅酶之间 碳负离子“中间体”。使用标准动力学方法、同位素探针、 和替代辅酶（3,4-二甲基-5-(2-焦磷酸乙基)噻唑鎓 离子，DMT）在存在和不存在类似物的情况下 硫胺素的氨基嘧啶基部分（AP）（4-氨基-2-甲基嘧啶，AMP）， (i) 去除两个功能之间的共价键的效果 硫胺素结构域和(ii)去除游离硫胺素上的AP部分 羟醛型加成-消除反应的能量-反应坐标图 将确定由丙酮酸脱羧酶催化的反应。这 信息将用于估计 PDC 结合的 DMT 中 C(2)-H 的 pKa， 确定 C(2)-叶立德和 C(α) 负碳离子/烯胺是否具有 在存在和 缺少 AMP，并评估 AP 部分结合的贡献 来激活酶的催化作用。二、绑定机制 硫胺素与大肠杆菌周质硫胺素结合蛋白将被检查为 使用标准的硫胺素依赖性酶的特别简单的模型 动力学和核磁共振方法以及同位素探针以确定是否结合 TN 很重要。