XAS STUDIES OF COPPER MONOOXYGENASE ENZYMES

铜单加氧酶的 XAS 研究

• 批准号: 7954249
• 负责人: Ninian J Blackburn
• 金额: $ 0.27万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7954249
• 关键词: Active Sites; Binding; Computer Retrieval of Information on Scientific Projects Database; Copper; Dependence; Dioxygen; Dopamine-beta-monooxygenase; Electrons; Enzymes; Exhibits; Funding; Grant; Institution; Measurement; Methionine; Mixed Function Oxygenases; Proteins; Reducing Agents; Research; Research Personnel; Resources; Roentgen Rays; Role; Source; Structure; Testing; United States National Institutes of Health; Water; absorption; base; expectation; mutant; peptidylglycine alpha-amidating monooxygenase; structural biology; synchrotron radiation

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. X-ray absorption spectroscopic measurements will be used to investigate the structure and mechanism of copper containing monooxygenase enzymes including peptidylglycine monooxygenase and dopamine beta monooxygenase. Absorption edge and EXAFS at the Cu K-edge will be combined with other spectroscopic approaches to provide critical tests of mechanism. A proposal that the active copper-dioxygen species is a Cu(II)-superoxo entity will be tested from the expectation that such a species should be EPR silent yet exhibit an oxidized absorption edge. The essential role of a coordinated methionine residue will be investigated through XAS studies of M314X, X= I, H, C, and D mutants. Detailed studies on the pH dependence will be carried out to test the hypothesis that the active site base is a coordinated water molecule bound at the CuM center. Finally, studies of reduction of the protein with a variety of reductants will probe mechanisms of electron entry and redistribution between the copper centers.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 X射线吸收光谱测量将用于研究含铜单加氧酶（包括肽基甘氨酸单加氧酶和多巴胺β单加氧酶）的结构和机制。 Cu K 边缘的吸收边和 EXAFS 将与其他光谱方法相结合，以提供关键的机制测试。活性铜双氧物质是 Cu(II)-超氧实体的提议将根据这样的物质应该是 EPR 沉默但表现出氧化吸收边缘的预期进行测试。将通过 M314X、X= I、H、C 和 D 突变体的 XAS 研究来研究协调蛋氨酸残基的重要作用。将进行对 pH 依赖性的详细研究，以检验活性位点碱基是结合在 CuM 中心的配位水分子的假设。最后，用各种还原剂还原蛋白质的研究将探讨电子进入和铜中心之间重新分布的机制。