RELATIONSHIP BETWEEN ENZYME STABILITY & ENZYME FUNCTION

酶稳定性之间的关系

• 批准号: 6188551
• 负责人: CARLOS N PACE
• 金额: $ 3.72万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6188551
• 关键词: active sites; bacterial proteins; binding sites; conformation; endoribonucleases; enzyme activity; enzyme inhibitors; enzyme structure; gene mutation; globular protein; microcalorimetry; molecular site; mutant; nuclear magnetic resonance spectroscopy; protein folding; thermodynamics; thermostability

We propose a collaborative project between Texas A and M University and the Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, in Moscow in the area of the conformational stability of enzymes. Specifically, we plan to test the hypothesis that protein residues contributing to catalysis are not optimal for protein stability, using microbial ribonucleases as models. We will study the effect of changes in the active site residues that abolish or diminish the enzyme's catalytic activity on the enzyme's thermostability using the RNases from Bacillus intermedius, binase, and Streptomyces aureofaciens, RNase Sa. In addition, we will estimate the stabilizing effect of restricting the mobility of particular residues in the RNase active sties by assessing the melting of proteins in complexes with the protein inhibitor barstar and its mutants. The primary goal of the investigation is to ascertain the existence in RNases of an interrelation between the thermostability of the protein and the conformational flexibility of active site residues required for catalytic efficiency. The Moscow group has recently observed an inverse dependence of enzymatic activity on thermal stability for chimeric RNases composed of different parts of barnase and binase molecules. The significance of the proposed project is that we will learn to predict how substitutions at active site residues will affect the function, folding, and stability of an enzyme. This can have future potential for redesign of enzymes to generate the desired catalytic properties and stability.

我们建议德克萨斯a和M大学与俄罗斯科学院恩格尔哈特分子生物学研究所在莫斯科开展酶构象稳定性领域的合作项目。具体来说，我们计划以微生物核糖核酸酶为模型，验证有助于催化的蛋白质残基不是蛋白质稳定性的最佳假设。我们将利用中间芽孢杆菌（Bacillus intermedius）、二酶（binase）和金黄色链霉菌（Streptomyces aureofaciens）的RNase Sa来研究活性位点残基的变化对酶的热稳定性的影响。此外，我们将通过评估与蛋白质抑制剂barstar及其突变体复合物中蛋白质的熔化来评估限制RNase活性位点中特定残基的流动性的稳定效果。研究的主要目的是确定rna酶中蛋白质的热稳定性和催化效率所需的活性位点残基的构象灵活性之间存在相互关系。莫斯科研究小组最近观察到，由巴纳酶和二酶分子的不同部分组成的嵌合rna酶的酶活性与热稳定性呈负相关。该项目的意义在于，我们将学会预测活性位点残基的取代如何影响酶的功能、折叠和稳定性。这可以为酶的重新设计提供未来的潜力，以产生所需的催化性能和稳定性。