Ru(II)-Containing Wires for ET Studies in Metalloenzymes

用于金属酶 ET 研究的含 Ru(II) 线

• 批准号: 7168001
• 负责人: Edith C Glazer
• 金额: $ 2.87万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7168001
• 关键词: Active Sites; Address; Affinity; Binding; Biological Assay; Catalysis; Chemicals; Complex; Complex Mixtures; Crystallography; Development; Disease; Disputes; Effectiveness; Electron Transport; Electronics; Environment; Enzymes; Fluorescence; Fluorescent Probes; Goals; Health; Lasers; Link; Mechanics; Mediating; Medical; Nitric Oxide; Nitric Oxide Synthase; Oxidases; Oxidation-Reduction; Play; Production; Proteins; Pteridines; Pterins; Regulation; Research; Role; Screening procedure; Specificity; Techniques; analog; assay development; base; cofactor; design; high throughput screening; inhibitor/antagonist; interest; metalloenzyme; progenitor; tetrahydrobiopterin; tool

DESCRIPTION (provided by applicant): The goal of this project is to use sensitizer-linked-substrates (SLS's) to probe the mechanism and mechanics of electron transfer-mediated catalysis in nitric oxide synthase (NOS). Part I involves the design and synthesis of pterin-based SLS's for NOS; both the natural cofactorthat is endogenously bound by the enzyme will be incorporated, as well as aromatic analogues which will serve as fluorescent probes of the pterin binding pocket. In Part II, using the catalytic oxidase domain of iNOS (iNOSoxy), the binding affinities of the pterin probes will be determined, fluorescence techniques will be used to explore structural and electronic perturbations near the active site during catalysis, x-ray crystallography will be utilized to elucidate the structural effects of incorporation of the probe into the protein, and finally, electron transfer studies will be employed to study the mechanism of NOS catalysis, especially the role of the pterin cofactor. In Part III, we will explore the development of these SLS's for use in high-throughput binding assays for NOS inhibitors. Finally, we will determine the efficacy of these NOS specific probes for studies on NOS in complex mixtures of other proteins.

描述（由申请人提供）：该项目的目的是使用敏化剂链接 - 覆盖物（SLS）来探测一氧化氧化物合酶（NOS）中电子转移介导的催化的机理和力学。第一部分涉及基于翼龙的SLS的设计和合成；两种天然辅助因子都将被酶内源性结合，以及芳香类似物，这些类似物将用作翼蛋白结合袋的荧光探针。在第二部分中，使用iNOS的催化氧化酶结构域（无毒），将确定翼龙探测的结合性，将使用荧光技术来探索在催化过程中的结构和电气扰动附近的结构和电气扰动，将利用X射线晶体学的机制来研究该概率的结构效应。 NOS催化，尤其是翼龙辅因子的作用。在第三部分中，我们将探索这些SLS的开发，以用于NOS抑制剂的高通量结合测定法。最后，我们将确定这些NOS特异性探针在其他蛋白质复杂混合物中对NOS的研究的疗效。