Redesigning Trypsin Via Mutagenesis

为了深入了解底物特异性，催化 蛋白水解酶突变体胰蛋白酶的结构与作用机制 将使用当前的蛋白质工程方法来产生。 两种一般方法用于改变酶：1）靶向 替换，然后分析变体酶2）区域 特定的取代与遗传选择筛选相结合。 在 每种情况下，胰蛋白酶的突变体都是根据结构产生的。 和/或功能原理，以1）理解特异性， 酶的催化能力，2）利用我们的理解来改变 以可预测的方式控制酶的活性。 变体 酶的设计，分离和纯化，其动力学 与天然酶相比。 选择的突变体是 然后选择更详细的动力学，理论和三维 结构分析 初步修改的结果将 为将来引入氨基酸变化提供了基础。 酵素 初步实验成功地解决了 特定氨基酸在底物识别和催化中的作用 酶的作用机制，并利用这些信息来改变酶的活性。 使用设计的金属结合位点的酶活性。 提出 实验涉及活性位点残基的氨基酸置换 用于涉及催化机制和底物的研究 识别. 通过计算机建模设计变体胰蛋白酶 基于我们目前对胰蛋白酶和/或其结构的理解， 以及与相关酶的功能关系。 方法 也是 开发用于筛选变异胰蛋白酶的大型文库， 遗传选择和噬菌体展示技术。 %%% 这些研究将有助于我们了解 胰蛋白酶和相关酶的结构/功能关系 丝氨酸蛋白酶 此外，这些研究将有助于提供一个 酶的从头设计的框架。//

To provide insight into the substrate specificity, catalytic mechanism and structure of a proteolytic enzyme, mutant trypsins 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 2) region specific substitutions coupled with a genetic selection\screen. In each case, mutants of trypsin are generated according to structural and/or functional principles to 1) understand the specificity and catalytic power of the enzyme and 2) use our 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 mutants are then chosen for more detailed kinetic, theoretical and 3-D structure analysis. Results from the initial 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 and in using that information to alter the enzyme activity using designed metal binding sites. Proposed experiments involve amino acid replacements of active site residues for studies involving the catalytic mechanism and substrate recognition. Variant trypsins are designed by computer modeling based on our current understanding of trypsin and/or its structural and functional relationship to related enzymes such. Methods are also being developed to screen large libraries of variant trypsins using genetic selections and bacteriophage display technologies. %%% These studies will contribute to our understanding of structure/function relationships in the enzyme trypsin and related serine proteases. In addition, these studies will help provide a framework for the de novo design of enzymes. //