RELATIONSHIP BETWEEN ENZYME STABILITY & ENZYME FUNCTION

我们提出了德克萨斯农工大学和莫斯科俄罗斯科学院恩格尔哈特分子生物学研究所在酶构象稳定性领域的合作项目。 具体来说，我们计划使用微生物核糖核酸酶作为模型来测试以下假设：有助于催化的蛋白质残基对于蛋白质稳定性而言并非最佳。 我们将使用来自中间芽孢杆菌 (Bacillus intermedius) 的 RNase、binase 和金黄色链霉菌 (Streptomyces aureofaciens) 的 RNase Sa，研究活性位点残基的变化对酶热稳定性的影响，这些变化会消除或降低酶的催化活性。 此外，我们将通过评估与蛋白质抑制剂 barstar 及其突变体形成的复合物中蛋白质的熔解来估计限制 RNase 活性粒细胞中特定残基移动性的稳定效果。 该研究的主要目标是确定核糖核酸酶中蛋白质的热稳定性与催化效率所需的活性位点残基的构象灵活性之间是否存在相互关系。 莫斯科小组最近观察到，由芽孢杆菌RNA酶和双糖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，我们将学习预测活性位点残基的取代将如何影响酶的功能、折叠和稳定性。 这可能具有重新设计酶以产生所需的催化特性和稳定性的潜力。

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.