XAS STUDIES OF BINUCLEAR METAL SITES IN PROTEINS OF ALKALINE PHOSPHATASE SUPERFA

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 磷酰基转移反应在广泛的生物过程中发挥着重要作用，如基础代谢，基因表达和细胞信号传导，催化磷反应的酶具有一些已知的最大速率加速。理解生物磷酰基转移化学的一个重大挑战是定义磷酰基转移酶实现其化学选择性的机制。例如，来自E. coli（AP）和X. axonopodis（NPP）都在由His和Asp残基连接的结构非常相似的双核锌活性位点催化磷酸二酯和单酯的水解反应。然而，AP优先催化单酯水解的因子为10^9，而NPP优先催化二酯水解的因子范围为10^2至10^6。有人认为，精细的结构差异的双核锌网站的AP和NPP是超出了目前的X射线晶体结构的分辨率（1.7？2.0 A）可以解释磷酸单酯或二酯的一些特异性。我们将使用XAS来研究AP和NPP中的双核活性中心的各种蛋白质形式，如全酶，底物-（磷酸单酯或二酯），抑制剂-（无机磷酸盐或磷酸单酯），和过渡态类似物（钒酸盐）结合的酶。这些研究将包括野生型含锌AP和NPP以及它们的钴取代类似物。通过在Zn，Co和V K边缘应用XAS技术，我们将能够在催化循环期间提供金属中心的电子和高分辨率结构信息。当耦合到生化和动力学数据，这些实验将有助于AP和NPP的比较结构分析，并推进我们的理解如何结构特征有助于双核金属网站的化学选择性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Phosporyl transfer reactions play a fundamental role in a wide range of biological processes such as basic metabolism, gene expression, and cell signaling, and enzymes that catalyze reactions at phosphorus have some of the largest rate accelerations known. A significant challenge in understanding the chemistry of biological phosphoryl transfer is in defining mechanisms through which phosphoryl transfer enzymes achieve their chemical selectivity. For example, alkaline phosphatase from E. coli (AP) and nucleotide pyrophosphatase/phosphodiesterase from X. axonopodis (NPP) both catalyze hydrolysis reactions of phosphate diesters and monoesters at a structurally very similar binuclear zinc active site ligated by His and Asp residues. However, AP preferentially catalyzes monoester hydrolysis by a factor of 10^9, whereas NPP preferentially catalyzes diester hydrolysis by factors ranging from 10^2 to 10^6. It has been suggested that fine structural differences between the binuclear Zn sites of AP and NPP that are beyond the resolution of the current x-ray crystallographic structures (1.7 ? 2.0 A) may account for some of the specificity for phosphate monoesters or diesters. We will use XAS to study the binuclear active center in AP and NPP for a variety of protein forms such as holoenzyme, substrate- (phosphate monoester or diester), inhibitor- (inorganic phosphate or phosphate monoester), and transition state analog (vanadate) bound enzymes. These studies will include wild-type zinc-containing AP and NPP as well as their cobalt-substituted analogs. By applying XAS technique at the Zn, Co, and V K-edge, we will be able to provide electronic and high-resolution structural information on the metal center during the catalytic cycle. When coupled to biochemical and kinetic data, these experiments will contribute to comparative structural analysis of AP and NPP and advance our understanding of how structural features contribute to chemical selectivity of the binuclear metal site.