Redesigning Trypsin via Mutagenesis

为了深入了解胰蛋白酶的底物特异性、催化机制和结构，将利用现有的分子遗传学、生物化学和生物物理学方法来生成和分析突变型胰蛋白酶。一般有三种改变酶的方法：1)靶向取代，2)区域特异性取代和3)自然适应分析。在每种情况下，胰蛋白酶的定点突变体都是根据结构和/或功能原理产生的。这种新型胰蛋白酶随后在细菌中过度表达。分离纯化了感兴趣的变异蛋白，并将其参数与天然酶进行了比较。然后选择突变体进行更详细的动力学，分子动力学和协调的三维结构分析。初步修饰的结果将为今后在酶中引入氨基酸修饰提供基础。最初的实验已经成功地解决了特定氨基酸在底物识别中的作用和酶的催化机制。拟议的实验包括用于研究催化机制的活性位点残基的氨基酸替代，底物结合袋残基和表面环残基。这些研究将有助于我们了解胰蛋白酶和相关丝氨酸蛋白酶的结构/功能关系。此外，这些研究将有助于为酶的从头设计提供一个框架。

To provide insight into the substrate specificity, catalytic mechanism and structure of trypsin, current methods of molecular genetics, biochemistry and biophysics will be used to generate and analyze mutant trypsins. Three general approaches are used to alter the enzyme: 1) targeted substitutions, 2) region specific substitutions and 3) natural adaptation analysis. In each case site-directed mutants of trypsin are generated according to structural and/or functional principles. The novel trypsins are then over-expressed in bacteria. The variant proteins of interest are isolated, purified and their parameters compared with the native enzyme. Selected mutants are then chosen for more detailed kinetic, molecular dynamic and coordinated three-dimensional structure analysis. Results from the initial modifications will provide a basis for introducing future amino aid changes into the enzyme. Initial experiments have been successful in addressing the role of specific amino acids in substrate recognition and the catalytic mechanism of the enzyme. Proposed experiments involve amino acid replacements of active site residues for studies of the catalytic mechanism, substrate binding pocket residues and surface loop residues. 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.