Interconversion of Specificity within Enzyme Families

酶家族内特异性的相互转换

• 批准号: 6859727
• 负责人: GEORGE Georgiou GEORGIOU
• 金额: $ 44.68万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6859727
• 关键词: biotechnology; chimeric proteins; chymotrypsin; combinatorial chemistry; dihydrofolate reductase; elastases; enzyme activity; enzyme structure; enzyme substrate; flow cytometry; glutathione transferase; protein engineering; protein folding; protein sequence; site directed mutagenesis

DESCRIPTION (provided by applicant): Understanding how function and substrate specificity are diversified within the members of an enzyme superfamily represents a key question in protein chemistry with profound implications for evolution, protein design and for the engineering of useful enzymes for biomedical applications. Here we present a comprehensive and highly integrated experimental research program for exploring how to interconvert substrate specificity among enzyme superfamily members exhibiting a low degree of amino acid sequence identity. As part of this work, entirely new chimeric enzymes, derived from the combinatorial assembly of subdomains from parental sequences, will be isolated from highly diverse libraries. Enzyme isolation and the interrogation of the libraries for function and substrate specificity will be accomplished by virtue of quantitative, ultra-high throughput screening that capitalizes predominantly on single cell, flow cytometric assays. Chimeric enzymes exhibiting desired profiles of catalytic activity and substrate selectivity will be crystallized, high resolution structures will be obtained where possible and finally, the catalytic mechanism of the enzymes will be analyzed in detail. As part of this study we will examine how the combinatorial assembly of protein subdomains can be employed to interconvert the specificity of serine proteases (elastase and chymotrypsin) and to transform the specificity of the human glutathione S transferase to that of the rat enzyme. In parallel we will explore the limits imposed by the decreasing amino acid sequence identity for the two parental genes selected for combination and seek to overcome these limits by mutagenesis. The gene pairs will be chosen from the extensive family of dihydrofolate reductase sequences allowing a systematic variation from 42% to 28% sequence identity--in all cases below that of classical DNA shuffling. Consequently, the proposed studies will help delineate the secondary structural elements and specific amino acids that dictate: (a) the cleavage specificity in trypsin proteases; (b) recognition of electrophile substrates in glutathione conjugation by GST enzymes and finally (c) protein folding and catalytic activity in dihydrofolate reductase. The generation of enzymes having novel substrate specificity profiles distinct from either parent will also be investigated. Finally, but perhaps equally importantly, this work will validate a unique, highly interdisciplinary approach for the exploration and deeper understanding of enzyme function.

描述（由申请人提供）：了解功能和底物特异性如何在酶超家族的成员中多样化，代表了蛋白质化学的关键问题，对进化，蛋白质设计以及对生物医学应用的有用酶的工程产生了深远的影响。在这里，我们提出了一项全面且高度集成的实验研究计划，用于探索如何在表现出低度氨基酸序列身份的酶超家族成员之间互换底物特异性。作为这项工作的一部分，将从高度多样化的文库中隔离出源自子序列的组合组合的全新嵌合酶。酶的隔离以及库的功能和底物特异性的询问将通过定量，超高的吞吐量筛选来实现，该筛选主要利用单个细胞，流式细胞仪测定。表现出所需的催化活性和底物选择性谱的嵌合酶，将在可能的情况下获得高分辨率结构，最后将详细分析酶的催化机理。作为这项研究的一部分，我们将研究如何使用蛋白质亚域的组合组合来互换丝氨酸蛋白酶（弹性酶和胰凝乳蛋白酶）的特异性，并转化人谷胱甘肽的转移酶的特异性与大鼠酶的特异性。同时，我们将探讨通过选择用于组合的两个父母基因的氨基酸序列身份所施加的限制，并试图通过诱变克服这些极限。该基因对将从二氢叶酸还原酶序列的广泛家族中进行选择，从而使从42％到28％的序列身份进行系统变化 - 在所有低于经典DNA取消的情况下。因此，拟议的研究将有助于描述决定：（a）胰蛋白酶蛋白酶中的裂解特异性； （b）识别GST酶在谷胱甘肽结合中的电生底物，最后（C）蛋白质折叠和二氢叶酸还原酶中的蛋白质折叠和催化活性。还将研究具有与任何父母不同的新型底物特异性谱的生成。最后，但也许同样重要的是，这项工作将验证一种独特的，高度的跨学科方法，以探索和更深入地了解酶功能。