RIBONUCLEASE SUPERFAMILY--STRUCTURE-FUNCTION STUDIES

本项目致力于获取详细的结构功能 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(RNaseA)，毒素，如人类嗜酸性粒细胞来源 神经毒素，人血管生成素等生长因子，以及蛋白质P-30 对人肿瘤细胞系有显著毒性。该序列 所有这些蛋白质的同源性和核糖核溶解活性 它们具有共同的催化机理和三级结构。这个 核糖核酸酶的大量结构和生化信息 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所展示的活动。核酸酶 A将被系统地突变为这些基因的初级序列 同源物，杂交蛋白将通过X射线进行检查 结晶学、动力学和适当的生物测定。 在每一种情况下，生物活动都与 核糖核溶解活性。因此，理解 详细的酶作用机理(S)所用的各种同源物。在……里面 除了解开酶活性和 生物学功能，杂交蛋白的生产将允许 对酶学有重要意义的现象的研究。 具体地说，核糖核酸酶超家族的成员利用结合 延长底物的能量，以提高催化效率高达 四个数量级。此外，催化反应的速度 超家族对氨基酸的特性高度敏感，这些氨基酸是 不直接参与化学转化或底物 绑定：kcat/kM跨越超家族的六个数量级。 远离位点的相互作用如何影响酶的活性 化学是酶学研究中的一个突出问题，也是 具有普遍重要性。 杂交核糖核酸酶的生产将允许对 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，这些相同的研究将 解决了“远距离效应”的根本问题 催化作用。确定负责的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了基础 发展。

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.