Biophysical Studies of Metalloenzymes

金属酶的生物物理研究

• 批准号: 0316038
• 负责人: Brian Hoffman
• 金额: $ 59.68万
• 依托单位: Northwestern University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-15 至 2008-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0316038&HistoricalAwards=false
• 关键词: Biophysical; Studies; Metalloenzymes

Electron paramagnetic resonance spectroscopy has long been a central tool for determining the structure and function of metalloenzyme active sites. This project will continue to develop unique methods to perform CW/pulsed ENDOR and ESEEM experiments at X (9 GHz) and Q (35 GHz) bands, and W band (95 GHz) microwave frequencies. This constellation of approaches, combined with new procedures developed for the analysis of frozen-solution ENDOR and ESEEM spectra, provides a powerful, selective, and sensitive tool for the characterization of metalloenzyme active sites and their interactions with substrates/inhibitors/products, both in the resting state and in key intermediates. This project will emphasize three important topics:(I) Localization of substrate/inhibitor binding at the molybdenum-iron cofactor of the nitrogenase MoFe protein and characterization of intermediate states formed during substrate reduction;(II) The enzymic mechanism of three members of "radical S-adenosylmethionine" superfamily, which all use [4Fe-4S] clusters and S-adenosylmethionine (AdoMet) to generate catalytically essential radicals.(III) Reactive intermediates and substrate/inhibitor interactions with molybdenum and tungsten oxotransferases (molybdopterins).Broader Impact: This project will develop sophisticated analytical procedures and algorithms for determining the complete coordination geometry and bonding at a metal center. These capabilities disseminated to the metallobiochemistry researchers via web, through formal collaborations and by informal consultation. Besides these research contributions, the project will provide research opportunities for a faculty member from an undergraduate institution. In addition, the project will involve training at the graduate and postdoctoral level.This project is funded jointly by Molecular Biochemistry and Molecular Biophysics.

长期以来，电子顺磁共振波谱一直是确定金属酶活性中心结构和功能的重要工具。该项目将继续开发独特的方法，在X(9 GHz)和Q(35 GHz)频段以及W频段(95 GHz)微波频率上进行连续波/脉冲ENDOR和ESEEM实验。这一系列方法与为分析冷冻溶液Endor和ESEEM光谱而开发的新程序相结合，为表征金属酶活性中心及其与静止状态和关键中间体中的底物/抑制剂/产物的相互作用提供了一个强大、选择性和灵敏的工具。这个项目将强调三个重要的主题：(I)固氮酶MoFe蛋白质的钼-铁辅助因子上底物/抑制剂结合的定位以及底物还原过程中形成的中间态的表征；(Ii)“自由基S-腺苷蛋氨酸”超家族三个成员的酶机制，它们都使用[4Fe-4S]簇和S-腺苷蛋氨酸(ADOMet)来产生催化必需自由基。(Iii)活性中间体以及底物/抑制剂与钼和钨氧转移酶(钼多酚)的相互作用。广泛影响：这个项目将开发复杂的分析程序和算法来确定金属中心的完整配位几何和成键。这些能力通过网络、正式合作和非正式协商传播给矿物生物化学研究人员。除了这些研究贡献，该项目还将为本科院校的教职员工提供研究机会。此外，该项目将包括研究生和博士后水平的培训。该项目由分子生物化学和分子生物物理学联合资助。