ROLES OF SODIUMK+ AND STRONG H-BONDS IN PROTEIN FUNCTION

钠和强氢键在蛋白质功能中的作用

• 批准号: 2910323
• 负责人: MICHAEL F DUNN
• 金额: $ 13.35万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2910323
• 关键词: allosteric site; bioenergetics; chemical kinetics; cofactor; conformation; enzyme complex; enzyme mechanism; enzyme structure; fluorescent dye /probe; hydrogen bond; isozymes; nuclear magnetic resonance spectroscopy; potassium ion; pyridoxal phosphate; sodium ion; tryptophan synthase

DESCRIPTION: Long Term Objectives. Elucidate fundamental principles of catalytic efficiency, allosteric regulation and substrate channeling in enzyme catalysis. Specific Aims. The work to be undertaken involves studies of the pyridoxal phosphate-requiring bacterial enzyme tryptophan synthase to accomplish the following: (a) Determine the mechanism of action of monovalent cations (MVC) in catalysis and regulation. (b) Establish a detailed mechanism for the roles of structural elements in the transmission of allosteric signals between subunits of the bienzyme complex. (c) Determine the catalytic function of protons thought to be involved in low-barrier H-bonds (LBHBs). Background. The tryptophan synthase bienzyme complex (alpha2-beta2) catalyzes the last two steps in the synthesis of L-Trp. Catalysis is intimately related to allosteric signaling and metabolite transfer between the alpha and beta sites. Reactions at the beta sites are hypothesized to switch alpha2-beta2 between low and high activity forms and "open" and "closed" conformations. This prevents the escape of the common metabolite, indole, and ensures that the alpha and beta reaction occur in phase. The binding of MVCs to a specific site (separate from the catalytic sites) activates specific steps in the catalytic pathway of tryptophan synthase. The MVC effect is hypothesized to result from selective lowering of the activation energies of certain steps through stabilizing interactions between the MVC and protein conformations complementary to the corresponding activated complexes. MVC binding also is essential to allosteric communication. Isotope effects suggest the involvement of an LBHB in tryptopha synthase catalysis. LBHBs are hypothesized to be strong bonds (12 to 24 kcal/mol) which lower activation energies, or stabilize conformation states. Methods. These hypotheses will be tested via the use of rapid kinetics, isotop effects, 1H NMR, mutants enzymes, and fluorescent probes. Significance to Huma Health and Disease. This work will contrbute new knowledge of structure-function relationships in enzyme catalysis and regulation concerning (a) The mechanism of action of MVCs. (b) The roles of conformation change in catalysis, allosteric regulation, and substrate channeling. (c) The roles, if any, of LBHBs in protein function.

描述：长期目标。 阐明的基本原则 催化效率，变构调节和底物通道 酶催化。 具体目标。 要进行的工作涉及 吡ido醇磷酸盐的细菌酶色氨酸的研究 合成酶完成以下操作：（a）确定作用机理 催化和调节中的单价阳离子（MVC）。 （b）建立 结构元素在传输中作用的详细机制 生物酶复合物亚基之间的变构信号。 （C） 确定被认为参与的质子的催化功能 低障碍H键（LBHBS）。 背景。 色氨酸合酶生物酶 复合物（alpha2-beta2）催化了合成的最后两个步骤 l-trp。 催化与变构信号和 α和β位点之间的代谢物转移。 Beta的反应 假设站点以在低活动和高活动之间切换α2-beta2 形式和“开放”和“封闭”构象。 这样可以防止逃脱 常见的代谢物，吲哚和确保α和β反应 在相位发生。 MVC与特定位点的结合（与 催化位点）在催化途径中激活特定步骤 色氨酸合酶。 MVC效应被认为是由 通过选择性降低某些步骤的激活能量 MVC和蛋白质构象之间的稳定相互作用 互补与相应的活化复合物。 MVC结合也是 变构沟通至关重要。 同位素效应表明 LBHB参与色氨酸合酶催化。 lbhbs是 假设是强键（12至24 kcal/mol），降低了激活 能量或稳定构象状态。 方法。 这些假设会 通过使用快速动力学，同位素效应，1H NMR，突变体进行测试 酶和荧光探针。 对Huma健康和疾病的意义。 这项工作将构成结构功能关系的新知识 在酶催化和调节中有关（a）的作用机理 MVC。 （b）构象变化在催化，变构中的作用 调节和底物通道。 （c）LBHB的角色（如果有的话） 蛋白质功能。