Bioinorganic Chemistry of Carbon Monoxide Dehydrogenase

一氧化碳脱氢酶的生物无机化学

• 批准号: 6610939
• 负责人: PAUL A. LINDAHL
• 金额: $ 27.65万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6610939
• 关键词: Mossbauer spectrometry; X ray crystallography; acetyl coA; active sites; carbon monoxide; catalyst; electron nuclear double resonance spectroscopy; electron spin resonance spectroscopy; enzyme activity; enzyme complex; freeze etching; iron sulfur protein; mass spectrometry; metal complex; nuclear magnetic resonance spectroscopy; oxidation reduction reaction; protein structure function; stop flow technique

DESCRIPTION (provided by applicant): The objective of this project is to elucidate the catalytic mechanism of CO dehydrogenase (a.k.a. acetyl-CoA synthase - ACS), emphasizing the structure and function of the unique Ni-Fe-S clusters contained therein. The enzyme is an alpha-2-beta-2 tetramer that catalyzes two reactions; the reversible reduction of CO2 to CO and the synthesis of acetyl-CoA. The former occurs at one Ni-Fe-S cluster in the beta subunit, while the latter reaction occurs at another Ni-Fe-S site in alpha. A tunnel through which CO migrates connects the two sites. The reaction mechanism is "organometallic" in nature; how does Ni promote this within an aqueous protein matrix? Mechanistic steps occurring at the two sites are synchronized, making it an attractive system to investigate details of metabolic channeling and active-site coupling. ACS is useful biotechnologically; organisms containing it reduce atmospheric levels of CO and degrade TNT. ACS is found in Clostridium difficile, a pathogenic organism responsible for the deaths of about 2000 people annually. Given the complexity of this enzyme, the general strategy used in this project will be to study each activity separately, and then compare observed properties with those of the bifunctional enzyme; such an approach may reveal some of the complexities of channeling and catalytic coupling. This approach is possible because isolated recombinant alpha subunits able to catalyze the synthesis of acetyl-CoA can be prepared, as can a homolog of the beta subunit that is able to catalyze CO/CO2 redox. Many of these developments (recombinant biosynthesis of active alpha, discovery of the tunnel and of active site coupling) arose from efforts of the previous granting period which resulted in 11 publications. The methodologies used for the project include enzyme kinetics (stopped-flow, rapid freeze-quench, and steady-state), spectroscopy (EPR, Mossbauer, and XAS), and X-ray diffraction. A rapid-freeze-quench instrument that allows samples to be prepared under reliably anaerobic conditions has been constructed, and should be an improvement over existing technology. Kinetic data will be simulated, ultimately leading to a comprehensive mechanistic model describing the general kinetic behavior of the enzyme. Crystals of alpha have been obtained, suggesting that an X-ray diffraction structure may be achievable. These studies require repetitive purification of at least 5 different proteins; funding for a preparative FPLC/HPLC is requested.

描述（由申请人提供）：该项目的目的是阐明CO脱氢酶（又名乙酰辅酶A合酶-ACS）的催化机制，强调其中包含的独特的Ni-Fe-S簇的结构和功能。该酶是一种 α-2-β-2 四聚体，可催化两个反应； CO2 可逆还原为 CO 和乙酰辅酶 A 的合成。前者发生在β亚基中的一个Ni-Fe-S簇上，而后者发生在α亚基中的另一个Ni-Fe-S位点上。 CO 迁移通过的隧道连接两个站点。反应机理本质上是“有机金属”； Ni 如何在水性蛋白质基质中促进这一作用？两个位点发生的机械步骤是同步的，这使其成为研究代谢通道和活性位点耦合细节的有吸引力的系统。 ACS 在生物技术上很有用；含有它的生物体可以降低大气中二氧化碳的含量并降解 TNT。 ACS 存在于艰难梭菌中，这是一种每年导致约 2000 人死亡的病原微生物。考虑到这种酶的复杂性，该项目使用的总体策略是分别研究每种活性，然后将观察到的特性与双功能酶的特性进行比较；这种方法可能揭示沟道和催化耦合的一些复杂性。这种方法是可能的，因为可以制备能够催化乙酰辅酶A合成的分离的重组α亚基，以及能够催化CO/CO2氧化还原的β亚基的同系物。其中许多进展（活性 α 的重组生物合成、隧道的发现和活性位点偶联）都源于上一个资助期的努力，并产生了 11 篇出版物。该项目使用的方法包括酶动力学（停流、快速冷冻淬灭和稳态）、光谱学（EPR、穆斯堡尔和 XAS）和 X 射线衍射。已经建造了一种快速冷冻淬灭仪器，可以在可靠的厌氧条件下制备样品，并且应该是对现有技术的改进。将模拟动力学数据，最终形成描述酶的一般动力学行为的综合机械模型。已经获得了α晶体，这表明X射线衍射结构是可以实现的。这些研究需要重复纯化至少 5 种不同的蛋白质；请求为制备型 FPLC/HPLC 提供资金。