Bioorganic Chemistry of Carbon Monoxide Dehydrogenase

本项目的长期目标是了解乙酰辅酶A的催化机理。 热醋酸莫雷氏菌的合成酶/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，具有催化活性的形式 分离的α是二聚体而不是单体，有一个催化活性亚基和一个结构亚基。我们会 表征二聚体，包括通过X射线衍射，并确定亚基不对称性是否可以 被淘汰了。其次，我们将使用核磁共振和X射线衍射来确定甲基和乙酰基催化 中间体与A簇活性中心的近端Ni(Nip)结合。如果是这样的话，Nip将只是第三家 金属蛋白中心，支持这种不寻常的有机金属键。第三，我们将使用生物物理学 以及氧化还原化学，以确定NiP在催化过程中是否达到零价氧化状态。如果是的话， 这在生物学上是史无前例的。第四，α亚基可以以两种构象存在，并经历 在每个催化循环中的构象变化。我们将开发一种荧光(FRET)分析来检测 这些变化并决定了每个催化步骤中存在的构象。最后，我们将扩大 我们最近开发的乙酰-辅酶A合成动力学/数学模型包含了额外的机理 特征，如构象变化，pH效应，以及协同CO抑制。这样的模型 代表了测试酶机制的最严格的方法。 与公共卫生的关系：艰难梭菌含有该酶，可引起 抗生素相关性结肠炎、毒性巨结肠、肠道穿孔，甚至人类死亡。赤霉菌的孢子 新型梭状芽孢杆菌感染小鼠肿瘤的实验研究 只在肿瘤的厌氧环境中生长，杀死癌细胞，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起的代谢作用 ，并确定防止艰难梭菌在肠道内扩散的策略和 促进新城疫杆菌在癌细胞中的增殖。此外，ACS/CODH还可以从 并从废弃的军事地点降解TNT。急性冠脉综合征/胆碱脱氢酶参与C1代谢和 它包含一条通道，CO通过该通道迁移，影响代谢通道的领域。

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.