CO DEHYDROGENASE AND ACETOCLASTIC METHANOGENESIS IN METHANOSARCINA BARKERI

在对巴氏甲烷链霉菌的实验中，确定了 碳-碳需要完整和充满能量的细胞膜。 用碳同位素交换法测定乙酸乙酯的键断裂 二氧化物。尽管没有检测到广泛的同位素交换 红曲霉菌的可溶性制剂、产甲烷和甲基-SCoM的形成 在三磷酸腺苷和氢气存在下，很容易观察到醋酸盐。 体外产甲烷的速度取决于三磷酸腺苷和 醋酸盐浓度。产甲烷开始前的滞后时间是 不是因为甲基-SCoM生产缓慢，而是因为 甲基-SCoM还原为HSCoM所需步骤(S)的活化 和甲烷。产甲烷和甲基-SCoM的两个过程 辅酶A刺激醋酸酯的形成 表明乙酰-辅酶A是所需的乙酸酯的活化形式 用于产甲烷过程中的切割。开发了一种简便的方法 用于分析负责形成甲基-SCoM的酶 来自甲基化的皮质激素化合物和HSCoM。M.细胞。 生长在醋酸盐上的Barkeri表现出基本上相同的水平 这种酶作为细胞生长在甲醇上。这表明了一个共同的 乙酸乙酯和甲醇两条路径中的甲基转移步骤 新陈代谢。将甲基转移酶纯化为NEAR 均一性，并被发现是具有相对分子质量的单体 三万七千人。表现出对高亲和力(Km=0.086 mM)的亲和力， 而弱结合的甲钴胺(Km=13.5 mm)。金属离子 螯合剂对酶有抑制作用，但会使酶重新激活。 通过去除螯合剂或添加二价金属离子。 大豆产甲烷和甲基-SCoM萃取物的补充 带有纯化的一氧化碳脱氢酶(CODH)的乙酸酯和 甲基转移酶分别未能刺激这些 流程。这表明CODH和CODH的水平 甲基转移酶在测试的条件下饱和， 另一个未确定的步骤(S)主要是速率限制。

In experiments with Methanosarcina barkeri it was determined that an intact and energized cell membrane is required for carbon-carbon bond cleavage of acetate as measured by isotopic exchange of carbon dioxide. Although extensive isotopic exchange was not detected in soluble preparations, methanogenesis and methyl-SCoM formation from acetate were readily observed in the presence of ATP and hydrogen. The rate of in vitro methanogenesis was dependent upon both ATP and acetate concentrations. The lag before onset of methanogenesis was not due to a slow production of methyl-SCoM, but rather to activation of step(s) needed for reduction of methyl-SCoM to HSCoM and methane. Both processes of methanogenesis and methyl-SCoM formation from acetate were stimulated by coenzyme A which indicates that acetyl-CoA is the activated form of acetate required for cleavage in methanogenesis. A convenient method was developed for analysis of the enzyme responsible for formation of methyl-SCoM from a methylated corrinoid compound and HSCoM. Cells of M. barkeri grown on acetate exhibited essentially the same level of this enzyme as cells grown on methanol. This suggests a common methyl group transfer step in both pathways of acetate and methanol metabolism. The methyltransferase enzyme was purified to near homogeneity and was found to be monomeric with a molecular weight of 37,000. It showed a high affinity for HSCoM (Km = 0.086 mM), but weakly bound methylcobalamin (Km = 13.5 mM). Metal ion chelators inhibited the enzyme but reactivation was brought about by removal of the chelator or addition of divalent metal ions. Supplementation of extracts producing methane and methyl-SCoM from acetate with purified carbon monoxide dehydrogenase (CODH) and the methyltransferase, respectively, failed to stimulate these processes. This suggests that the levels of CODH and methyltransferase were saturating under the conditions tested and that another unidentified step(s) was predominantly rate limiting.