Glucose Signaling: Regulation of Enzyme Activity

葡萄糖信号传导：酶活性的调节

• 批准号: 6876505
• 负责人: DONALD W PETTIGREW
• 金额: $ 20.37万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6876505
• 关键词: Escherichia coli; Haemophilus influenzae; X ray crystallography; active sites; allosteric site; biochemical evolution; bioenergetics; biological signal transduction; chemical kinetics; directed evolution; enzyme activity; fluorescence microscopy; glucose metabolism; glycerol kinase; ligands; microorganism metabolism; protein structure function

DESCRIPTION (provided by applicant): Glucose controls utilization and metabolic fates of carbohydrates in prokaryotes and eukaryotes. Knowledge of molecular mechanisms of this control is important for understanding basic aspects of biology and many pathological states in humans. Protein-protein interactions are the basis for most biological regulation, including much of glucose signaling. This work focuses on protein-protein interactions in molecular mechanisms of allosteric control by which signals that are generated by glucose uptake and metabolism regulate carbon source metabolism and fate in bacteria. The studies focus on allosteric control of members of the sugar kinase/actin/hspTO superfamily of enzymes. A central issue is determining whether the motions of the layered structure of the conserved ATPase catalytic core of superfamily members, which are believed to be part of the normal catalytic cycle, are the basis for allosteric control. The issue is addressed by comparing the allosteric control of E. coli glycerol kinase by IIAGIc, a phosphotransferase system protein, to that of superfamily members that have gained IIAGIc control as a result of molecular evolution in the laboratory. Evolved enzymes that show a remarkable range of IIAGIc binding affinity and inhibition efficacy have been prepared. Additional enzymes will be evolved from the absolutely non-allosteric superfamily member, E. coli xylulokinase, by using combinatorial mutagenesis and molecular breeding methods. A powerful positive genetic selection will be used to identify the target enzymes from libraries of candidates. The energetic of thermodynamic coupling will be determined from steady state kinetics and ligand binding studies by using the approach of linked functions. The static and dynamic structures will be determined by X-ray crystallography and fluorescence methods. IIAGIc allosteric control of glycerol kinases displays behavior that is not seen for classical allosteric enzymes, activation with respect to substrate binding but inhibition with respect to enzyme catalysis. The energetics of such non-classical allosteric control has not been described. These studies will reveal the role of the conserved catalytic core structure and the active site closure conformational change in allosteric control of superfamily members and the molecular basis for glucose control of carbohydrate metabolism in E. coli.

描述（由申请人提供）： 葡萄糖控制原核生物和真核生物中碳水化合物的代谢命运。对该控制的分子机制的了解对于理解生物学的基本方面和人类的许多病理状态很重要。蛋白质蛋白相互作用是大多数生物学调节的基础，包括大部分葡萄糖信号传导。这项工作的重点是变构控制的分子机制中的蛋白质 - 蛋白质相互作用，通过这些相变的信号通过葡萄糖摄取和代谢产生的信号调节细菌中碳源代谢和命运。这些研究集中于糖激酶/肌动蛋白/hspto酶的成员的变构控制。一个核心问题是确定超家族成员保守ATPase催化核心的分层结构的运动（认为是正常催化循环的一部分）是变构控制的基础。通过将磷酸转移酶系统蛋白IIAGIC对大肠杆菌甘油激酶的变构控制与由于实验室分子进化而获得IIAGIC控制的超家族成员的变构控制。已经准备好表现出显着的IIAGIC结合亲和力和抑制功效的进化酶。通过使用联合诱变和分子育种方法，将从绝对非官方的超家族成员E. coli xylulokinase演化出其他酶。强大的积极遗传选择将用于识别候选人库中的目标酶。热力学耦合的能量将通过使用链接功能的方法从稳态动力学和配体结合研究确定。静态和动态结构将由X射线晶体学和荧光方法确定。甘油激酶的IIAGIC变构控制表现出对经典变构酶没有看出的行为，这是对底物结合的激活，但在酶催化方面抑制了。这种非古典变构控制的能量学尚未描述。这些研究将揭示保守的催化核心结构和主动部位闭合构象变化在超家族成员的变构控制中，以及大肠杆菌中碳水化合物代谢的葡萄糖控制的分子基础。