MECHANISMS OF PEPSIN CATALYSIS AND INHIBITION

描述（改编自申请人摘要）：本研究的目的 计划是为了发现观察到的独特特异性的起源， 由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，对于消化酶，到有限的， 对于参与激活单个激素原的酶。 因为所有 该家族的成员具有相似的三维结构， 催化机理，选择性抑制剂的设计必须基于 活动站点内差异的详细信息。 通过结合使用 动力学研究用合成寡肽底物，定点 活性位点裂缝内残基的诱变有助于 结合和特异性，以及家族内的比较， 将发现紧密、特异性结合的基本原理。 努力将 重点实现以下具体目标：1. 积极的研究的扩展 位点相互作用进入裂缝的主要区域。 2. 比较 人组织蛋白酶E的结构和性质与其他哺乳动物 酶，胃蛋白酶和组织蛋白酶D。3. 嵌合体的产生和分析 其中N-末端和C-末端结构域互换以 评估远程静电效应的影响。 4. 评价 蛔虫的三维结构与 胃蛋白酶抑制剂和独特的抑制特异性。 目标1将继续检查扩展的活性部位裂隙， 使胃蛋白酶组织蛋白酶D发生新的突变， 这将在新提案的目标2中详细研究。 目标3 提出了一个扩大的诱变策略，通过创造新的 蛋白质物种的整体静电环境的作用， 将探索点突变的混合背景。 在目标4中， 目的是通过获得蛋白质-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 蛔虫抑制剂强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.