MECHANISMS OF PEPSIN CATALYSIS AND INHIBITION

描述(改编自申请人的摘要)：本报告的目的 程序是为了发现观察到的独特特性的起源 天冬氨酸蛋白酶家族催化的蛋白质切割反应。 特异性的例子从广泛的消化酶到有限的， 对于参与激活单一激素原的酶。作为所有人 这个家族的成员有类似的三维结构和 催化机理、选择性缓蚀剂的设计必须以 活动站点内部差异的详细信息。通过结合使用 用于动力学研究的合成寡肽底物，定点定向 活性部位裂隙内残基的诱变作用 约束性和专一性，以及家族内部的比较，精确的 我们将发现紧密、特定绑定的基本原理。努力将会 重点抓好以下具体目标：1.拓展主动学习 位置相互作用进入裂隙的主要区域。2.中美两国比较 人组织蛋白酶E与其他哺乳动物的结构和性质 酶、胃酶和组织蛋白酶D.3.嵌合体的产生和分析 N-末端和C-末端结构域互换为 评估远程静电效应的影响。4.评估 蛔虫的三维结构与其形态结构的关系 胃蛋白酶抑制剂具有独特的特异性和抑制作用。 目标1将继续检查扩展的活性部位裂解 使胃酶、组织蛋白酶D和新的目标组织蛋白酶发生新的突变 E.将在新提案的目标2中对其进行详细研究。目标3 建议通过创建新的 蛋白质物种所处的整体静电环境和作用 我们将探索点突变的混合背景。在目标4中， 目的是通过获得以下信息来扩大蛋白质水平的研究 一种特定的天然蛋白质抑制剂的结构。分子 蛔虫抑制物强烈的胃蛋白酶导向特异性的起源 可能会被发现。

DESCRIPTION (Adapted from applicant's abstract): The objectives of this program are to discover the origin of the unique specificities observed in protein cleavage reactions catalyzed by the aspartic proteinase family. Examples of specificity range from broad, for digestive enzymes, to limited, for enzymes that participate in activation of a single prohormone. As all members of this family have similar three-dimensional structures and catalytic mechanisms, design of selective inhibitors must be based on details of the differences within the active sites. By combining the use of synthetic oligopeptide substrates for kinetic studies, site-directed mutagenesis of residues within the active site clefts contributing to binding and specificity, and comparisons within the family, the precise rationale for tight, specific binding will be discovered. Efforts will focus on the following specific aims: 1. Expansion of the study of active site interactions into the prime region of the cleft. 2. Comparison of the structure and properties of human cathepsin E with the other mammalian enzymes, pepsin and cathepsin D. 3. Generation and analysis of chimeric constructs in which N-terminal and C-terminal domains are interchanged to evaluate the effect of long-range electrostatic effects. 4. Evaluation of the relationship between the three-dimensional structure of the Ascaris pepsin inhibitor and the unique specificity of inhibition. Aim 1 will continue the examination of the extended active site cleft by making new mutations in pepsin, cathepsin D, and a new objective, cathepsin E, which will be studied in detail in aim 2 of the new proposal. Aim 3 proposes an expansion of a mutagenesis strategy through the creation of new protein species where the role of the overall electrostatic environment and mixed backgrounds for point mutations will be explored. In aim 4, the intent is to expand investigation at the protein-protein level by obtaining the structure of a specific natural protein inhibitor. The molecular origins of the strong pepsin directed specificity of the Ascaris inhibitor may be found.