CO DEHYDROGENASE AND ACETOCLASTIC METHANOGENESIS IN METHANOSARCINA BARKERI

巴克甲烷八球菌中的 CO 脱氢酶和乙酰分解产甲烷作用

• 批准号: 3966517
• 负责人: D A GRAHAME
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3966517
• 关键词: Methanobacteriaceae; carbon monoxide; ionic strengths

A study has been conducted on carbon monoxide dehydrogenase (CODH) from acetate-grown cells of Methanosarcina barkeri. Under conditions of high ionic strength the enzyme exists in an aggregated state along with a discrete set of other proteins (total mol. wt. approx. 3,000,000). Dissociation of the aggregate occurred when the ionic strength was decreased by dialysis. The disaggregated form of CODH retained full activity and exhibited a molecular weight of approximately 161,000. An efficient purification procedure was developed which produced high yields of pure CODH. The method consisted of the following steps which were carried out under strictly anaerobic conditions in the NIH Anaerobic Laboratory: 1. Isolation of the CODH aggregate by gel-filtration and subsequent dissociation; 2. Chromatography on Phenyl Sepharose; and 3. Hydroxylapatite chromatography. CODH appeared to be less hydrophobic than any of the other protein components of the aggregate. It bound tightly to hydroxylapatite and, thus, may have a relatively large number of exposed carboxylic acid residues. Pure CODH is composed of two subunits of molecular weights 93,800 and 20,400. Among various compounds tested, oxygen and cyanide are potent inactivators. Glyoxaldehyde inactivation occurred only during enzymatic turnover, which suggests that a reactive group is formed, or exposed, on an enzyme intermediate in catalysis.

对一氧化碳脱氢酶（CODH）进行了研究 巴克甲烷八叠球菌的醋酸盐生长细胞。 在高条件下 离子强度酶以聚集状态存在 离散的其他蛋白质组（总分子量约 3,000,000）。 当离子强度为 通过透析减少。 CODH 的分解形式保留完整 活性，分子量约为 161,000。 一个 开发了有效的纯化程序，产生了高产率 纯CODH。 该方法包括以下步骤： 在 NIH 厌氧实验室中严格厌氧条件下进行 实验室： 1. 通过凝胶过滤分离 CODH 聚集体 随后解离； 2. 苯基琼脂糖凝胶层析；和 3. 羟基磷灰石色谱法。 CODH 的疏水性似乎比 聚集体的任何其他蛋白质成分。 它紧紧地束缚在 羟基磷灰石，因此可能有相对大量的暴露 羧酸残基。 纯 CODH 由两个亚基组成 分子量为 93,800 和 20,400。 在测试的各种化合物中， 氧气和氰化物是有效的灭活剂。 乙二醛失活 仅在酶周转期间发生，这表明反应性 基团在催化过程中形成或暴露在酶中间体上。