DOMAINS OF HISTIDINE TRNA SYNTHETASE SUBSTRATE BINDING

组氨酸 TRNA 合成酶底物结合结构域

• 批准号: 6180629
• 负责人: CHRISTOPHER S FRANCKLYN
• 金额: $ 19.97万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180629
• 关键词: DNA footprinting; Escherichia coli; X ray crystallography; active sites; acylation; aminoacid tRNA ligase; analog; arginine; binding proteins; carboxylate; enzyme activity; enzyme complex; enzyme structure; enzyme substrate; histidine; magnesium; molecular genetics; mutant; nuclear magnetic resonance spectroscopy; protein biosynthesis; protein purification; site directed mutagenesis

DESCRIPTION: Accurate protein synthesis requires that each aminoacyl-TRNA synthetase must select its substrates from a pool of chemically similar molecules. The relationship between this discrimination and catalytic function is an important problem in biology. The applicant's experimental system uses structural biology, biochemistry, and molecular genetics to investigate the structure and function of E. coli histidyl-TRNA synthetase, one of the smallest and simplest class II aminoacyl-TRNA synthetases. Using this system, structures of enzyme-substrate and enzyme-product complexes have been obtained. In addition, mutants of histidyl-TRNA synthetase with altered TRNA recognition and other catalytically significant differences have been characterized and the minimal catalytic domain has been defined. Catalysis appears to be promoted by both "induced fit" binding which places substrates in conformations suitable for subsequent reaction, and by the use of binding energy to lower reaction barriers. These hypotheses will be tested by investigating structure-activity relationships through the application of pre-steady state kinetics to the reactions of wild-type and mutant enzymes. The proposed studies aim to: 1) define the basis of sequence specific TRNA recognition by X-ray crystallography and double mutant thermodynamic cycle analysis; 2) probe the essential features of the catalytic mechanism by investigating putative catalytic residues, the magnesium binding site, and the basis of amino acid selection; and 3) characterize the structure and function of the catalytically active N-terminal domain with respect to dimerization, TRNA binding, and specific kinetic defects. Among the aminoacyl-TRNA synthetases, HisRS aminoacylates a wide variety of RNA substrates, so these studies will be valuable for their contribution to understanding RNA recognition by proteins. Moreover, the HisRS catalytic domain is structurally related to translational regulatory proteins, and the human enzyme is a major antigen in a class of autoimmune diseases characterized by autoantibodies against protein:RNA complexes. If funded this project would replace the currently funded R29, whose aims have been to investigate the basis of substrate selection by E. coli histidyl-TRNA synthetase.

描述：准确的蛋白质合成需要每个氨酰基-TRNA 合成酶必须从化学相似的池中选择其底物 分子。 这种歧视和催化之间的关系 功能是生物学中的一个重要问题。 申请人的实验 系统利用结构生物学、生物化学和分子遗传学来 研究大肠杆菌组氨酰-TRNA 合成酶的结构和功能， 最小和最简单的 II 类氨酰基-TRNA 合成酶之一。 使用 该系统，酶-底物和酶-产物复合物的结构 已获得。 此外，组氨酰-TRNA 合成酶突变体 改变 TRNA 识别和其他催化显着差异 已经被表征并且最小催化域已经被定义。 催化似乎是通过“诱导契合”结合来促进的，这使得 底物的构象适合后续反应，并通过使用 结合能以降低反应势垒。 这些假设将 通过调查结构-活动关系进行测试 前稳态动力学在野生型和野生型反应中的应用 突变酶。 拟议研究的目的是：1) 定义序列特异性 TRNA 的基础 X射线晶体学和双突变热力学循环识别 分析; 2) 探究催化机制的本质特征 研究假定的催化残基、镁结合位点，以及 氨基酸选择的基础； 3）表征结构和 催化活性 N 端结构域的功能 二聚化、TRNA 结合和特定动力学缺陷。 在氨酰基-TRNA 合成酶中，HisRS 可氨酰化多种酶 RNA 底物，因此这些研究对于它们的贡献将是有价值的 了解蛋白质对 RNA 的识别。 此外，HisRS 催化 结构域在结构上与翻译调节蛋白相关，并且 人类酶是一类自身免疫性疾病的主要抗原 其特征是针对蛋白质：RNA 复合物的自身抗体。 如果有资助 该项目将取代目前资助的 R29，其目标是 研究大肠杆菌组氨酰-TRNA 底物选择的基础 合成酶。