ALLOSTERIC REGULATION OF SUBSTRATE CHANNELING

底物通道的变构调节

• 批准号: 7060882
• 负责人: MICHAEL F DUNN
• 金额: $ 18.95万
• 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 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7060882
• 关键词: Salmonella typhimurium; X ray crystallography; allosteric site; bioenergetics; cations; chemical binding; chemical kinetics; cofactor; conformation; enzyme mechanism; enzyme structure; enzyme substrate complex; mass spectrometry; nuclear magnetic resonance spectroscopy; protein structure function; tryptophan synthase

DESCRIPTION (provided by applicant): Lonq-Term Objectives: Elucidate fundamental relationships of chemical catalysis, enzyme conformational dynamics, allosteric regulation, and enzyme structure to the mechanism of metabolite transfer between enzyme pairs (substrate channeling) in a metabolic cycle. Specific Aims: To achieve a detailed mechanistic description of the allosteric regulation of substrate channeling in the bacterial tryptophan synthase bienzyme complex. The roles played by the binding of allosteric effectors, the formation of intermediates, and the binding of a monovalent cation cofactor in the regulation of channeling will be determined. Backqround: The tryptophan synthase bienzyme complex catalyzes the last two steps in the biosynthesis of L-tryptophan. Catalysis is intimately related to allosteric signaling and metabolite transfer between the alpha- and beta-sites. Hypotheses To Be Tested: (a) The alpha- and beta-subunits of the bienzyme complex switch between "open", "partially open" and "closed" conformation states during the catalytic cycle in response to binding and covalent reaction of substrates. (b) Certain covalent states of the beta-site give rise to a slow switching from the closed to the open conformation state(s) that is functionally important for substrate channeling. (c) Monovalent cation (MVC) binding plays a cofactor role that modulates the catalytic and conformational energetics of the complex. (d) The BetaAsp305-BeataArg141 salt bridge is critically important for the allosteric regulation and reaction specificity of the complex. Methods: The hypotheses will be tested via the use of rapid kinetics, equilibrium binding, 19F NMR, mutant enzymes, substrate analogues, optical spectroscopy and x-ray crystallography. The project Significance to Human Health and Disease derives from the new knowledge about structure-function relationships for the class of protein nanostructures consisting of enzyme-enzyme complexes that are organized into metabolic pathways. These nanostructures are subject to non-traditional forms of allosteric regulation that are poorly understood. Four project areas are of fundamental interest: (a) The roles of protein structural elements and protein conformational dynamics in substrate channeling between enzyme pairs in metabolic pathways. (b) The roles played by protein conformational dynamics in biological function, (c) the roles of monovalent cations in the modulation of protein function, (d) the role played by substrates as allosteric effectors in substrate channeling.

描述（由申请人提供）：LONQ期限目标：阐明化学催化，酶构象动力学，变构调节和酶结构的基本关系与代谢循环中酶对（底物通道）之间的代谢物转移机制。具体目的：实现细菌色氨酸合酶生物酶复合物中底物通道的变构调节的详细机械描述。将确定将确定变构效应子的结合，中间体的形成以及单价阳离子辅因子在通道调节中的结合所起的作用。后方：色氨酸合酶生物酶复合物催化L- tryptophan的生物合成的最后两个步骤。催化与α-和β点之间的变构信号传导和代谢产物转移密切相关。要测试的假设：（a）在催化循环中“开放”，“部分开放”和“封闭”构象之间的生物酶复合物切换的α-和β-亚基，这是响应于底物的结合和共价反应的响应。 （b）β地点的某些共价状态从闭合构象状态慢速切换，这对于底物通道功能至关重要。 （c）单价阳离子（MVC）结合起着调节复合物的催化和构象能量的辅助作用。 （d）betaasp305-beataarg141盐桥对于络合物的变构调节和反应特异性至关重要。方法：假设将通过使用快速动力学，平衡结合，19F NMR，突变酶，底物类似物，光谱和X射线晶体学测试。对人类健康和疾病的项目意义源自有关蛋白质纳米结构类别的结构功能关系的新知识，这些蛋白质纳米结构由酶 - 酶复合物组成，这些酶 - 酶复合物被组织成代谢途径。这些纳米结构受到较低理解的变构调节的非传统形式。四个项目领域具有基本兴趣：（a）蛋白质结构元件和蛋白质构象动力学在代谢途径中酶对之间底物引导中的作用。 （b）蛋白质构象动力学在生物学功能中所起的作用，（c）单价阳离子在蛋白质功能调节中的作用，（d）底物作为底物通道中的变构效应子的作用。