Bioorganic Chemistry of Carbon Monoxide Dehydrogenase

一氧化碳脱氢酶的生物有机化学

• 批准号: 7481583
• 负责人: PAUL A. LINDAHL
• 金额: $ 26.74万
• 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 2008-09-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7481583
• 关键词: Acetyl Coenzyme A; Active Sites; Antibiotic-Associated Colitis; Binding; Bioinorganic Chemistry; Biological; Biological Assay; Biology; Biophysics; Cancerous; Carbon; Carbon monoxide dehydrogenase; Catalysis; Cells; Cessation of life; Chemistry; Clostridium; Clostridium difficile; Cobalamin; Coenzyme A; Complement component C1s; Complex; Condition; Corrinoids; Couples; Data; Dependence; Development; Dimerization; Electrochemistry; Electron Nuclear Double Resonance; Environment; Enzymes; Equation; Exhibits; Film; Fluorescence; Fluorescence Resonance Energy Transfer; Funding; Heterogeneity; Human; Intestinal Perforation; Intestines; Ions; Iron; Kinetics; Metabolic; Metabolism; Metalloproteins; Metals; Microscopic; Military Personnel; Modeling; Molecular Conformation; Mossbauer Spectroscopy; Mus; NMR Spectroscopy; Nature; Object Attachment; Oxidation-Reduction; Property; Prosthesis; Protein Conformation; Proteins; Public Health; Rate; Reaction; Reagent; Reducing Agents; Reporter; Reproduction spores; Roentgen Rays; Role; Role playing therapy; Sampling; Signal Transduction; Simulate; Site; Solutions; Structure; Sulfur; Survival Rate; System; Testing; Tetrapyrroles; Time; Toxic Megacolon; Transition Elements; United States National Institutes of Health; X ray diffraction analysis; X-Ray Diffraction; absorption; base; chemical reaction; dimer; enzyme activity; enzyme mechanism; killings; mathematical model; methyl group; model design; monomer; mutant; novel; oxidation; pathogen; planetary Atmosphere; potentiometric titration; prevent; tumor

The long-term objective of this project is to understand the catalytic mechanism of acetyl-CoA synthase/carbon monoxide dehydrogenase (ACS/CODH) from Moorella thermoacetica. We have five specific aims for the next four years. First, we have recently discovered that the catalytically active form of isolated alpha is a dimer not a monomer, with 1 catalytically active subunit and 1 structural subunit. We will characterize the dimer, including by X-ray diffraction, and determine whether subunit asymmetry can be eliminated. Second, we will use NMR and X-ray diffraction to determine whether methyl and acetyl catalytic intermediates bind to the proximal Ni (Nip) of the A-cluster active site. If so, Nip would be only the third metalloprotein center known to support such unusual organometallic bonds. Third, we will use biophysics and redox chemistry to determine whether Nip achieves a zero-valent oxidation state during catalysis. If so, this would be unprecedented in biology. Fourth, the alpha subunit can exist in 2 conformations and undergo a conformational change during each catalytic cycle. We will develop a fluorescence (FRET) assay to sense these changes and determine which conformation is present at each catalytic step. Finally, we will expand our recently developed kinetic/mathematical model of acetyl-CoA synthesis to include additional mechanistic features, such as the conformational changes, pH effects, and cooperative CO inhibition. Such models represent the most rigorous means of testing enzyme mechanisms. Relavance to Public Health: Clostridium difficile, which contains this enzyme ACS/CODH, causes antibiotic-associated colitis, toxic megacolon, intestinal perforations and even death in humans. Spores of Clostridium novyi-NT, which almost certainly contain ACS/CODH, injected into cancerous tumors of mice grow exclusively in the anaerobic environment of the tumor, killing cancerous cells and increasing survival rates. Our mechanistic study of ACS/CODH will help define the metabolic roles played by the same enzymes in these pathogens, and identify strategies for preventing the proliferation of C. difficile in intestines and for encouraging the proliferation of C. novyi in cancerous cells. Also, ACS/CODH removes CO from the atmosphere and degrades TNT from abandoned military sites. ACS/CODH is involved in C1 metabolism and it contains a tunnel through which CO migrates, impacting the field of metabolic channeling.

该项目的长期目标是了解乙酰辅酶A的催化机制 来自热乙酸穆尔氏菌的合成酶/一氧化碳脱氢酶 (ACS/CODH)。我们有五个 未来四年的具体目标。首先，我们最近发现催化活性形式 分离的α是二聚体而不是单体，具有1个催化活性亚基和1个结构亚基。我们将 表征二聚体，包括通过 X 射线衍射，并确定亚基不对称是否可以 被淘汰。其次，我们将使用NMR和X射线衍射来确定甲基和乙酰基是否催化 中间体与 A 团簇活性位点的近端 Ni (Nip) 结合。如果是这样，Nip只是第三个 已知金属蛋白中心支持这种不寻常的有机金属键。第三，我们将利用生物物理学 和氧化还原化学以确定 Nip 在催化过程中是否达到零价氧化态。如果是这样， 这在生物学中是前所未有的。第四，α亚基可以以2种构象存在并经历 每个催化循环期间的构象变化。我们将开发一种荧光 (FRET) 检测方法来检测 这些变化并确定每个催化步骤中存在哪种构象。最后我们来扩展一下 我们最近开发的乙酰辅酶A合成动力学/数学模型包括额外的机制 特征，例如构象变化、pH 效应和协同 CO 抑制。此类车型 代表了测试酶机制的最严格的方法。 与公共健康的相关性：含有 ACS/CODH 酶的艰难梭菌会导致 抗生素相关结肠炎、中毒性巨结肠、肠穿孔甚至人类死亡。孢子 将几乎肯定含有 ACS/CODH 的 Clostridium novyi-NT 注射到小鼠的癌性肿瘤中 仅在肿瘤的厌氧环境中生长，杀死癌细胞并提高存活率 费率。我们对 ACS/CODH 的机制研究将有助于确定相同酶所发挥的代谢作用 在这些病原体中，并确定防止艰难梭菌在肠道中增殖的策略以及 促进癌细胞中诺氏梭菌的增殖。此外，ACS/CODH 还可去除 CO2 大气层并降解废弃军事基地的 TNT。 ACS/CODH 参与 C1 代谢 它包含一个二氧化碳迁移的隧道，影响代谢通道领域。