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

碱性磷酸酶SUPERFA蛋白质中双核金属位点的XAS研究

• 批准号: 8169935
• 负责人: KEITH O HODGSON
• 金额: $ 0.03万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169935
• 关键词: Acceleration; Accounting; Active Sites; Alkaline Phosphatase; Binding; Biochemical; Biochemistry; Biological Process; Chemicals; Cobalt; Computer Retrieval of Information on Scientific Projects Database; Coupled; Data; Electronics; Enzymes; Escherichia coli; Funding; Gene Expression; Grant; Holoenzymes; Hydrolysis; Institution; Kinetics; Metabolism; Metals; Nucleotide pyrophosphatase; Phosphorus; Play; Proteins; Reaction; Research; Research Personnel; Resolution; Resources; Role; Signal Transduction; Site; Source; Specificity; Structure; Techniques; United States National Institutes of Health; Vanadates; Zinc; analog; comparative; inhibitor/antagonist; inorganic phosphate; phosphoric diester hydrolase; research study

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 在广泛的生物学过程中，例如碱性代谢，基因表达和细胞信号传导，促磷酸反应的酶具有一些最大的速率加速度，已知的磷酸化反应在诸如碱性代谢，基因表达和细胞信号传导等广泛的生物学过程中起着基本作用。理解生物磷酸化学转移化学的重大挑战是定义机制，磷酸化酶可以实现其化学选择性。例如，来自大肠杆菌（AP）和核苷酸焦磷酸酶/磷酸二酯酶的碱性磷酸酶来自X.轴突（NPP）均催化磷酸盐diesters和单植物的水解反应，在结构上非常相似的双核ZINC活性位点在结构上非常相似，而他的ASP残留物则与他的ASP残留物相连。然而，AP优先用10^9催化单酯的水解，而NPP优先通过范围为10^2到10^6的因素优先催化二酯的水解。有人提出，AP和NPP的双核Zn位点之间的细节结构差异超出了当前X射线晶体学结构的分辨率（1.7？2.0 a），可能会说明磷酸盐单植物或二耶斯特人的某些特异性。我们将使用XAS研究AP和NPP中的双核活性中心，以了解多种蛋白质形式，例如全酶，底物 - （磷酸单磷酸单酯或二酯），抑制剂 - （磷酸磷酸或磷酸单磷酸单酯）和过渡状态（氧化氢）的糖（氧化氢）。这些研究将包括含锌锌的AP和NPP及其钴取代的类似物。通过在Zn，Co和V K-Edge上应用XAS技术，我们将能够在催化循环期间在金属中心提供电子和高分辨率的结构信息。当与生化和动力学数据耦合时，这些实验将有助于对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位点的化学选择性的理解。