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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 磷酸转移反应在基础代谢、基因表达和细胞信号等广泛的生物过程中发挥着重要作用，而催化磷反应的酶具有已知的一些最大的速度加速。理解生物磷酰转移化学的一个重大挑战是定义磷酰转移酶实现其化学选择性的机制。例如，来自大肠杆菌的碱性磷酸酶(AP)和来自X.axonopodis的核苷酸焦磷酸/磷酸二酯酶(NPP)都在由His和Asp残基连接的结构非常相似的双核锌活性部位催化磷酸二酯和单酯的水解反应。然而，AP优先催化单酯水解率为10^9，而NPP优先催化双酯水解率为10^2~10^6。有人认为，AP和NPP的双核锌中心之间的细微结构差异超出了当前X射线晶体结构(1.7？2.0A)的分辨率，这可能是导致磷酸单酯或双酯的一些特殊性的原因。我们将使用XAS研究AP和NPP中各种蛋白质形式的双核活性中心，如全酶、底物-(磷酸单酯或双酯)、抑制剂-(无机磷酸或磷酸单酯)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。通过在锌、钴和钒的K-边应用XAS技术，我们将能够在催化循环中提供关于金属中心的电子和高分辨率结构信息。结合生化和动力学数据，这些实验将有助于AP和NPP的比较结构分析，并促进我们对结构特征如何有助于双核金属中心的化学选择性的理解。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.