Engineering the Function of Serine Proteases

丝氨酸蛋白酶功能的工程改造

• 批准号: 9604379
• 负责人: Charles Craik
• 金额: $ 28.1万
• 依托单位: University of California-San Francisco
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-01 至 2002-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9604379&HistoricalAwards=false
• 关键词: Engineering; Function; Serine; Proteases

96-04379 Craik Part 1. Technical The study is to provide insight into the substrate specificity, catalytic mechanism, and structure of the chymotrypsin family of serine proteases. Variant enzymes will be generated using current methods of protein engineering. Two general approaches are used to alter the enzyme: (1) targeted substitutions followed by analysis of the variant enzyme; and (2) random substitutions coupled with an in vitro affinity selection. In each case, variants of serine proteases are generated according to structural and/or functional principles to understand the specificity and catalytic power of the enzyme and use this understanding to alter the activity of the enzyme in a predictable fashion. Variant enzymes are designed, isolated, and purified, and their kinetic parameters compared with the native enzyme. Selected variants are then chosen for more detailed kinetic, theoretical, and three-dimensional structure analysis. Results form the inital modifications will provide a basis for introducing future amino acid changes into the enzyme. Initial experiments have been successful in addressing the role of specific amino acids in substrate recognition and catalytic mechanism of the enzyme trypsin and in using that information to alter the enzyme activity using designed metal binding sites and substrate assisted catalysis. Other experiments involve amino acid replacements for studies including substrate recognition and metalloregulation. Variant proteases are designed by computer modeling based on our current understanding of proteases including fiddler crab collagenase, granzymes, and other related members of this family. These studies will contribute to our understanding of structure/function relationships in serine proteases. In addtion, these studies will help develop unique specificities for peptide and protein hydrolysis. Part 2. non-technical The relationship between the three dimensional structure and the function of an enzyme is still poorly understood. This is particularly evident in the field of protein engineering where efforts are made to alter the activity of a protein in a predictable fashion. Macromolecular recognition and the basis of catalytic power in an enzyme remain unexplained in terms of basic general principles that can be applied to a protein catalyst. This study will address these two fundamental questions as a long term goal by studying the contributions of certain amino acids in the substrate specificity and catalytic mechanism of serine protease. The aim is to be achieved by creating and analyzing altered protease which contribute to our understanding of mechanisms involved in ligand binding and bond making and bonding breaking. The altered enzyme will be generated using current methods of protein engineering as designed by computer modeling based on our current knowledge of proteases of this family. These studies will contribute to our understanding of structure/function relationships in protein degradative enzymes. ***

96-04379 Craik第1部分技术本研究旨在深入了解丝氨酸蛋白酶的凝乳酶家族的底物专一性、催化机制和结构。变异酶将使用目前的蛋白质工程方法产生。一般使用两种方法来改变酶：(1)靶向替换，然后分析变异酶；(2)随机替换结合体外亲和力选择。在每种情况下，根据结构和/或功能原理产生丝氨酸蛋白酶的变体，以了解酶的特异性和催化能力，并利用这种理解以可预测的方式改变酶的活性。设计、分离和纯化了变异酶，并与天然酶的动力学参数进行了比较。然后选择选定的变体进行更详细的动力学、理论和三维结构分析。初始修饰的结果将为将来在酶中引入氨基酸变化提供基础。最初的实验已经成功地解决了特定氨基酸在底物识别和酶胰酶的催化机制中的作用，并利用这些信息通过设计的金属结合部位和底物辅助催化来改变酶的活性。其他实验包括氨基酸替代研究，包括底物识别和金属调节。变异蛋白水解酶是根据我们目前对河蟹胶原酶、颗粒酶等相关家族成员的理解，通过计算机模拟来设计的。这些研究将有助于我们理解丝氨酸蛋白酶的结构/功能关系。此外，这些研究将有助于开发多肽和蛋白质水解的独特特性。第二部分：非技术的酶的三维结构和功能之间的关系仍然知之甚少。这一点在蛋白质工程领域尤为明显，在该领域，人们努力以可预测的方式改变蛋白质的活性。根据可应用于蛋白质催化剂的基本一般原理，酶中的大分子识别和催化能力的基础仍未得到解释。这项研究将通过研究某些氨基酸在丝氨酸蛋白酶底物专一性和催化机制中的作用来解决这两个基本问题作为长期目标。目的是通过创造和分析改变的蛋白酶来实现的，这有助于我们理解涉及配体结合、键形成和键断裂的机制。改变后的酶将使用目前的蛋白质工程方法，通过计算机模拟设计，基于我们目前对这个家族的蛋白酶的知识。这些研究将有助于我们理解蛋白质降解酶的结构/功能关系。***