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）：本项目的目标是使用敏化剂连接的底物（SLS）来探测一氧化氮合酶（NOS）中电子转移介导的催化的机制和力学。第一部分涉及的设计和合成的蝶呤为基础的SLS的NOS的天然cofactorthat是内源性结合的酶将被纳入，以及芳香族类似物，这将作为荧光探针的蝶呤结合口袋。在第二部分中，使用iNOS的催化氧化酶结构域（iNOSoxy），将确定蝶呤探针的结合亲和力，将使用荧光技术来探索催化期间活性位点附近的结构和电子扰动，将使用X射线晶体学来阐明探针掺入蛋白质的结构效应，最后，电子转移研究将用于研究NOS催化的机制，特别是蝶呤辅因子的作用。在第三部分中，我们将探讨这些SLS的发展用于高通量结合测定NOS抑制剂。最后，我们将确定这些NOS特异性探针在其他蛋白质的复杂混合物中对NOS的研究的功效。