MECHANISM OF THE STAPHYLOCOCCAL NUCLEASE REACTION

葡萄球菌核酸酶是由金黄色葡萄球菌产生并催化 DNA和RNA的水解以产生3‘-单核苷酸。核酸酶是 一种具有很好的结构特征的酶，它特别容易服从 对其结构和化学机理的成因和核磁共振研究 作用：该酶在一个线性序列中只含有149个氨基酸 通过蛋白质和DNA的氨基酸序列分析建立 克隆基因的序列分析；克隆基因的三维结构 该酶已被测定为1.5A分辨率；而该基因 酶在大肠杆菌中得到了高水平的表达 几种表达载体。我们之前已经调查了几个 酶催化的化学机制的一些方面 DNA和RNA的水解酶。在这份提案中，我们描述了一个全面的 引物定向定点突变技术在进一步研究中的应用 研究活性部位中存在的一些氨基酸的作用 酶的活性。特别是，我们计划探索羧酸盐的作用 存在于残基43(野生型酶中的谷氨酸)，氨基 推测影响底物与酶结合的酸(赖氨酸， 精氨酸和酪氨酸与阴离子磷酸盐和 形成碱基结合位点的其他残基)，以及氨基酸 被推定为结合催化所需的必需钙。这些属性 在这项研究中产生的突变酶的部分将进行动力学分析 并通过X射线结晶学和核磁共振波谱对其结构进行了表征。 虽然X射线结晶学将在其他地方进行，但核磁共振 研究将在我们的实验室进行，并将制定方法学 为了简化核酸酶SO的核磁共振波谱性质 质子化学位移，质子-质子耦合常数，以及 质子-质子核Overhaser效应可以用来推断信息 关于核酸酶野生型和突变型的构象。

Staphylococcal nuclease is produced by Staphylococcus aureus and catalyzes the hydrolysis of DNA and RNA to yield 3'-mononucleotides. The nuclease is a structurally very well characterized enzyme that is particularly amenable to genetic and NMR studies of its structure and chemical mechanism of action: the enzyme contains only 149 amino acids in a linear sequence established by both amino acid sequence analysis of the protein and DNA sequence analysis of the cloned gene; the three dimensional structure of the enzyme has been determined to 1.5 A resolution; and the gene for the enzyme has been expressed at high levels in Escherichia coli by the use of several expression plasmids. We have previously investigated several aspects of the chemical mechanism by which the enzyme catalyzes the hydrolysis of DNA and RNA. In this proposal we describe a comprehensive application of primer directed site specific mutagenesis to further investigate the role of a number of amino acids present in the active site of the enzyme. In particular, we plan to probe the role of the carboxylate group present at residue 43 (glutamate in the wild type enzyme), the amino acids presumed to effect binding of substrate to the enzyme (lysines, arginines, and tyrosines that interact with the anionic phosphates and other residues that form the base binding site), and the amino acids presumed to bind the essential Ca2+ required for catalysis. The properties of the mutant enzymes generated in this study will be analyzed kinetically and structurally by both X-ray crystallography and by NMR spectroscopy. While the X-ray crystallography will be carried out elsewhere, the NMR studies will be conducted in our laboratory and will develop methodology for the simplification of the NMR spectral properties of the nuclease so that proton chemical shifts, proton-proton coupling constants, and proton-proton nuclear Overhauser effects can be used to deduce information about the conformations of wild type and mutant forms of the nuclease.