RIBONUCLEASE SUPERFAMILY--STRUCTURE-FUNCTION STUDIES

核糖核酸酶超家族——结构-功能研究

• 批准号: 2187731
• 负责人: STEVEN C. ALMO
• 金额: $ 18.63万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2187731
• 关键词: X ray crystallography; active sites; angiogenesis factor; antineoplastics; chemical kinetics; crystallization; cyclic nucleoside monophosphate; cytosine nucleotides; deoxyribonucleotides; dinucleotide; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate analog; enzyme substrate complex; hybrid enzyme; laboratory rabbit; ligands; mutant; neurotoxins; pancreatic ribonuclease; protein structure function; site directed mutagenesis; tissue /cell culture

This project is concerned with obtaining detailed structure-function correlations for members of the ribonuclease superfamily. This family of proteins includes digestive enzymes such bovine pancreatic ribonuclease (RNase A), toxins, such as human eosinophil derived neurotoxin, growth factors such as human angiogenin, and P-30, a protein with significant toxicity against human tumor cell lines. The sequence homology and ribonucleolytic activity of all these proteins, suggests that they share a common catalytic mechanism and tertiary structure. The vast amount of structural and biochemical information available for RNase A provides a unique opportunity to examine the basis for the biological activities displayed by members of the ribonuclease superfamily. RNase A will be systematically mutated towards the primary sequence of these homologs, and the hybrid proteins will be examined by X-ray crystallography, kinetics and the appropriate bioassays. In every case, the biological activities have been linked to the ribonucleolytic activity. It is therefore crucial to understand the detailed enzymatic mechanism(s) used by the various homologs. In addition to unraveling the connection between enzymatic activity and biological function, the production of hybrid proteins will allow for the study of phenomena which are of fundamental importance to enzymology. Specifically, members of the ribonuclease superfamily utilize the binding energy of extended substrates to enhance catalytic efficiency by up to four orders of magnitude. Furthermore, the rate of catalysis in the superfamily is highly sensitive to the identity of amino acids which are not directly involved in either the chemical transformation or substrate binding: kcat/Km spans six orders of magnitude across the superfamily. How enzymatic activity is effected by interactions distant from the site of chemistry is one of the outstanding questions in enzymology, and is of general importance. The production of hybrid ribonucleases will allow for an examination of important biological activities such as neurotoxicity, growth factor activity and tumor cytotoxicity. In addition, these same studies will address the fundamental question of 'long range distant effects' in catalysis. Defining the structural and chemical determinants responsible for these biological activities will be instrumental in understanding their underlying mechanism and in providing a basis for therapeutic development.

本项目涉及获得详细的结构-功能 核糖核酸酶超家族成员的相关性。 这个家庭 包括消化酶，如牛胰腺 核糖核酸酶（RNA酶A）、毒素，如人嗜酸性粒细胞来源的 神经毒素，生长因子如人血管生成素，和P-30，一种蛋白质 对人肿瘤细胞系具有显著的毒性。 序列 所有这些蛋白质的同源性和核糖核酸溶解活性，表明 它们具有共同的催化机制和三级结构。 的 大量的结构和生物化学信息可用于核糖核酸酶 A提供了一个独特的机会来检查生物学的基础， 核糖核酸酶超家族成员显示的活性。 rna酶 A将被系统地突变为这些的一级序列， 同源物，杂交蛋白将通过X射线检查 晶体学、动力学和适当的生物测定。 在每一种情况下，生物活动都与 核糖核酸分解活性 因此，了解 各种同系物使用的详细酶机制。 在 除了解开酶活性和 生物功能，杂交蛋白的产生将允许 研究对酶学具有根本重要性的现象。 具体地，核糖核酸酶超家族的成员利用结合蛋白质。 能量的扩展基板，以提高催化效率高达 四个数量级。 此外，催化剂中的催化速率 超家族对氨基酸的身份高度敏感， 不直接参与化学转化或底物 结合：kcat/Km跨越整个超家族的六个数量级。 酶活性如何受到远离位点的相互作用的影响 化学反应是酶学中的一个突出问题， 具有普遍重要性。 杂交核糖核酸酶的产生将允许检查 重要的生物活性，如神经毒性、生长因子 活性和肿瘤细胞毒性。 此外，这些研究将 解决“远距离效应”的基本问题， 催化作用 确定结构和化学决定因素 因为这些生物活动将有助于理解 它们的潜在机制，并为治疗提供基础 发展