DOMAINS OF HISTIDINE TRNA SYNTHETASE SUBSTRATE BINDING

组氨酸 TRNA 合成酶底物结合结构域

• 批准号: 2910284
• 负责人: CHRISTOPHER S FRANCKLYN
• 金额: $ 19.39万
• 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 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2910284
• 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：准确的蛋白质合成要求每个氨基酰基-TRNA 合成酶必须从化学相似的池中选择其底物 分子。 这种歧视与催化之间的关系 功能是生物学中的重要问题。 申请人的实验 系统使用结构生物学，生物化学和分子遗传学 研究大肠杆菌组基-TRNA合成酶的结构和功能， 最小，最简单的II类氨基酰基-TRNA合成酶之一。 使用 该系统，酶 - 基底和酶产生络合物的结构 已获得。 另外，与组合基-TRNA合成酶的突变体与 改变tRNA识别和其他催化显着差异 已经表征并定义了最小的催化结构域。 催化似乎是通过两个位置的“诱导拟合”结合来促进的 构象的底物适用于后续反应，并通过使用 与较低反应屏障的结合能。 这些假设将是 通过通过研究结构活性关系进行测试 将稳态的状态动力学应用于野生型的反应和 突变酶。 提出的研究目的是：1）定义序列特定tRNA的基础 X射线晶体学和双突变体热力学循环的识别 分析; 2）探测催化机制的基本特征 研究推定的催化残基，镁结合位点和 氨基酸选择的基础； 3）表征结构和 相对于催化活性N末端结构域的功能 二聚化，tRNA结合和特定动力学缺陷。 在氨基酰基-TRNA合成酶中，HISRS氨基酰基各种各样的种类繁多 RNA底物，因此这些研究对于它们对 了解蛋白质识别RNA。 而且，HISRS催化 域在结构上与翻译调节蛋白有关， 人酶是一类自身免疫性疾病的主要抗原 具有针对蛋白质的自身抗体的特征：RNA复合物。 如果资助 该项目将取代当前资助的R29，其目的是 研究大肠杆菌组基型tRNA的底物选择的基础 合成酶。