Physiological Substrates and Products of Carbon Monoxide Dehydrogenase from Methanosarcina barkeri

目前的证据表明， 乙酸，一氧化碳脱氢酶，来自甲烷八叠球菌 巴氏杆菌介导乙酰辅酶A裂解为甲基和羰基 中间体，然后氧化羰基部分以提供 用于将甲基CoM还原成甲烷的电子。 然而，已知分离的一氧化碳脱氢酶 仅催化甲基紫精连接的碳氧化 一氧化碳转化为二氧化碳。 这项研究将建立 一氧化碳的生理底物和产物 脱氢酶，并直接测试此建议的作用 产甲烷酶 已经开发了一种测定方法， 测量二氧化碳从乙酸盐中的释放， 加入接受甲基辅酶，如辅酶 M，其中甲基还原酶被抑制的提取物。 使用 该测定方法中甲基化的途径中间体将 通过其替代CoM的能力以及 放射性示踪剂研究。 甲基中间体接近于 乙酰辅酶A的裂解将用于监测 纯化能够切割乙酰辅酶A的蛋白， 甲基中间体和二氧化碳。 由乙酸盐形成甲烷和二氧化碳是一种 有机物生物降解的基本反应 在许多厌氧环境中。 醋酸盐是关键 在有机聚合物分解产生的中间体 生物体的生长和人类的工业和 农业活动。 乙酸是直接前体 生物产生的大部分甲烷 由微生物介导的反应。 一些微生物可以 在需要碳- 碳键断裂。 这种酶 生物学上重要的一步是这个主题 调查。*

Current evidence indicates that, during methanogenesis from acetate, carbon monoxide dehydrogenase from Methanosarcina barkeri mediates cleavage of acetylCoA to methyl and carbonyl intermediates, then oxidizes the carbonyl moiety to provide electrons for the reduction of methylCoM to methane. However, isolated carbon monoxide dehydrogenase is known to catalyze only the methylviologen linked oxidation of carbon monoxide to carbon dioxide. This research will establish the physiological substrates and products of carbon monoxide dehydrogenase, and directly test the proposed role of this enzyme in methanogenesis. An assay has been developed to measure carbon dioxide evolution from acetate dependent upon the addition of a methyl accepting coenzyme, such as coenzyme M, to extracts in which methylreductase is inhibited. Using this assay methylated intermediates of the pathway will be identified by their ability to replace CoM as well as by radiotracer studies. The methyl intermediate proximal to the cleavage of acetyl-CoA will then be used to monitor purification of proteins capable of cleaving acetyl-CoA to that methyl intermediate and carbon dioxide.%%% Formation of methane and carbon dioxide from acetate is an essential reaction in the bio-degradation of organic matter in many anaerobic environments. Acetate is a key intermediate in the breakdown of organic polymers generated both by the growth of organisms and by human industrial and agricultural activities. Acetic acid is the direct precursor of the majority of biologically generated methane in a reaction mediated by microorganisms. Some microorganisms can synthesize methane in a reaction requiring that the carbon- carbon bond be broken. The enzyme which carries out this biologically important step is the subject of this investigation.***