Engineering the Function of Serine Proteases

96-04379 第一部分：技术本研究旨在深入了解丝氨酸蛋白酶糜蛋白酶家族的底物特异性、催化机制和结构。变体酶将使用当前的蛋白质工程方法产生。 使用两种一般方法来改变酶：（1）靶向取代，然后分析变体酶;和（2）随机取代与体外亲和力选择偶联。 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，以理解酶的特异性和催化能力，并使用这种理解以可预测的方式改变酶的活性。 变异酶的设计，分离和纯化，并与天然酶的动力学参数进行比较。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将氨基酸变化引入酶提供基础。 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蛋白酶的催化机制中的作用，并使用设计的金属结合位点和底物辅助催化来使用该信息改变酶活性。 其他实验涉及氨基酸替代研究，包括底物识别和金属调节。 变体蛋白酶是基于我们目前对蛋白酶的理解通过计算机建模设计的，所述蛋白酶包括招潮蟹胶原酶、颗粒酶和该家族的其他相关成员。 这些研究将有助于我们了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肽和蛋白质水解的独特特异性。 部分2.酶的三维结构和功能之间的关系仍然知之甚少。 这在蛋白质工程领域尤其明显，其中努力以可预测的方式改变蛋白质的活性。 酶中的大分子识别和催化能力的基础仍然没有解释可以应用于蛋白质催化剂的基本一般原理。 本研究将解决这两个基本问题作为一个长期的目标，通过研究某些氨基酸在丝氨酸蛋白酶的底物特异性和催化机制的贡献。 其目的是通过创建和分析改变的蛋白酶，这有助于我们了解参与配体结合和键的形成和键断裂的机制来实现。改变的酶将使用当前的蛋白质工程方法产生，所述方法是基于我们对该家族蛋白酶的当前知识通过计算机建模设计的。 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。 ***

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. ***