MECHANISM OF THE STAPHYLOCOCCAL NUCLEASE REACTION

这个项目是为了了解金属的机制 葡萄球菌核酸酶催化DNA水解Ca ~（2+）的研究 （SNase）。 SNase特别适合于结构/功能的研究 由于蛋白质很小（149个氨基酸）， 该结构已经通过X射线以高分辨率确定 晶体学，和NMR共振（1H，13 C和15 N）几乎是 完全分配;此外，各种动力学工具可用于 研究了水解反应的机理。 我们已经删除了 在SNase的活性位点中的构象柔性Ω环（以 产生δ SNase），并设计了活性变体的代谢筛选 的SNASE。 我们将使用基于盒的基因随机诱变， 1）确定一般基础的几何要求 通过残基43处的羧酸酯基团（Glu和Asp）催化; 2）确定 咪唑鎓对一般碱催化的几何要求 基团（His）; 3）确定除了Ca以外的金属离子是否 2+能促进DNA的水解; 4）确定 杂环碱基特异性可以改变以有利于G和/或C， 比A和T更好。在特定目的1和2的情况下，随机诱变将 用于改变通过缺失 欧米茄环;在具体目标3和4的情况下，定点 诱变和随机诱变将一起使用，以改变 结合必需金属的官能团的身份和位置 离子和碱，从而可以改变酶的特异性。 所有 四个具体目标将涉及重要的生物物理（X射线和 NMR）表征突变酶的结构， 详细的动力学和机理的研究催化的反应， 突变酶

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.