STRUCTURAL ANALYSIS OF ENGINEERING TRYPSINS

蛋白水解酶的识别和催化作用在宿主中占有重要地位 医学上具有重要意义的生物过程。鲜为人知 关于在ATOM上识别酶-底物的细节 水平。我们将确定识别的结构机制 通过我们对胰蛋白酶、胶原酶、Ecotin的实验 和肠肽酶。胰腺蛋白水解酶，弹性蛋白酶， 凝乳酶和胰蛋白酶被认为是它们的底物。 从催化剂附近的特异性口袋中辨别 这些酶的机械。一种遥远的同系物，丝氨酸胶原酶， 具有与这些酶不同的结合部位，这些酶提供 显著的催化能力，降解胶原三螺旋，a 一种能抵抗所有胰腺蛋白酶的蛋白质。这个 胰酶原激活剂，肠肽酶，是 其目标序列Asp Lys具有惊人的特异性。 它的选择性与限制性内切酶不相上下。这些 胰腺和胶原酶被Ecotin和And抑制 来自大肠杆菌的282个氨基酸的不同寻常的蛋白质。这种蛋白质使用 未知的相互作用，以抑制所有这些蛋白酶。我们会 确定这些相互作用的原子水平机制，使用 蛋白质工程和X射线结晶学的工具。这 知识还可以用来理解 酶的专一性和速率增强，也可以在 设计可用作治疗或诊断的新型酶 探员们。

Recognition and catalysis by proteases figure prominently in a host of biological processes of medical importance. Little is known about the details of protease-substrate recognition at atomic level. We will determine structural mechanisms for recognition in protease through our experiments with trypsin, collagenase, ecotin and enteropeptidase. The pancreatic proteases, elastase, chymotrypsin and trypsin are thought to derive their substrate discrimination from a specificity pocket adjacent to the catalytic machinery of these enzymes. A distant homolog, serine collagenase, has binding sites distinct from these enzymes that provide the remarkable catalytic power to degrade the collagen triple helix, a protein that is resistant to all the pancreatic proteases. The activator of the pancreatic proenzymes, enteropeptidase, is strikingly specific for its target sequence, Asp Asp Asp Asp Lys. It rivals restriction endonucleases in its selectivity. These pancreatic and collagenolytic proteases are inhibited by ecotin, an unusual protein of 282 amino acids from E. coli. This protein uses unknown interactions to inhibit all of these proteases. We will determine the atomic level mechanisms of these interactions using the tools of protein engineering and X-ray crystallography. This knowledge can also be used to understand general mechanisms for enzyme specificity and rate enhancement and can also be valuable in designing novel enzymes that can serve as therapeutic or diagnostic agents.