MECHANISM OF THE STAPHYLOCOCCAL NUCLEASE REACTION

这个项目是为了了解金属的机制。 离子(钙)辅助金黄色葡萄球菌核酸酶催化DNA的水解 (SNase)。SNase特别适合于结构/功能的研究 催化关系由于蛋白质很小(149个氨基酸)， 用x射线衍射仪测定了该化合物的高分辨结构。 结晶学，核磁共振波谱(1H、13C和15N)几乎是 完全分配；此外，各种动力工具可用于 研究了该反应的反应机理。我们已经删除了 SNase(TO)活性部位的构象柔性欧米伽环 产生Delta SNase)，并设计了一种针对活动变体的代谢筛查 和SNase的关系。我们将使用基于盒子的随机基因突变来 Delta SNase to 1)确定通用底座的几何要求 43位残基上的羧酸基(谷氨酸和天冬氨酸)的催化作用；2)测定 咪唑类化合物一般碱催化的几何要求 43位基团(His)；3)确定除Ca以外的金属离子 2)能促进DNA的水解；4)确定 杂环碱基专一性可以改变为有利于G和/或C 在特定目标1和2的情况下，随机突变将 用来改变由缺失的 Omega循环；在具体目标3和4的情况下，现场定向 诱变和随机诱变将一起使用来改变 键合必需金属的官能团的结构和位置 离子和碱，这样酶的专一性就可以改变。全 四个具体目标将涉及重要的生物物理(x射线和 核磁共振)表征突变酶的结构 催化反应的详细动力学和机理研究 突变的酶。

This project is directed toward understanding the mechanism of the metal ion (Ca 2+) assisted hydrolysis of DNA catalyzed by Staphylococcal nuclease (SNase). SNase is particularly amenable to the study of structure/function relationships in catalysis since the protein is small (149 amino acids), the structure has been determined to high resolution by x-ray crystallography, and the NMR resonances (1H, 13C, and 15N) are almost totally assigned; furthermore, a variety of kinetic tools is available to study the mechanism of the hydrolysis reaction. We have already deleted a conformationally flexible Omega-loop in the active site of SNase (to produce delta SNase) and devised a metabolic screen for activity variants of SNase. We will use casette based random mutagenesis of the gene for delta SNase to 1) determine the geometric requirements for general base catalysis by a carboxylate group (Glu and Asp) at residue 43; 2) determine the geometric requirements for general base catalysis by an imidazolium group (His) at residue 43; 3) determine whether metals ions other than Ca 2+ can promote the hydrolysis of DNA; and 4) determine whether the heterocyclic base specificity can be altered to favor G and/or C rather than A and T. In the case of Specific Aims 1 and 2, random mutagenesis will be used to alter the conformation of the Beta-turn produced by deletion of the Omega-loop; in the case of Specific Aims 3 and 4, site-directed mutagenesis and random mutagenesis will be used together to alter the identity and positions of functional groups that bind the essential metal ion and base so that the specificity of the enzyme can be altered. All four Specific Aims will involve both significant biophysical (x-ray and NMR) characterization of the structures of mutant enzymes in addition to detailed kinetic and mechanistic studies of the reactions catalyzed by the mutant enzymes.