Purification and characterization of dimethylamine:5-hydroxybenzimidazolyl-cobamide methyltransferase from Methanosarcina barkeri Fusaro.
Purification and characterization of dimethylamine:5-hydroxybenzimidazolyl-cobamide methyltransferase from Methanosarcina barkeri Fusaro.
复制标题
巴克甲烷八叠球菌 Fusaro 中二甲胺：5-羟基苯并咪唑基-钴酰胺甲基转移酶的纯化和表征。
DOI：
--
复制
发表时间：
1998
期刊：
影响因子：
--
通讯作者：
G. D. Vogels
中科院分区：
文献类型：
--
作者：
R. Wassenaar;J. Keltjens;C. van der Drift;G. D. Vogels
二甲胺：5-羟基苯并咪唑基钴酰胺甲基转移酶 (DMA-MT) 从在三甲胺上生长的 Methanosarcina barkeri Fusaro 细胞中纯化。在甲基钴胺：辅酶 M 甲基转移酶同工酶 II [MT2(II)] 存在下，该酶非常特异性地催化二甲胺（表观 Km = 0.45 mM）和 2-巯基乙磺酸（辅酶 M）化学计量转化为单甲胺和甲基辅酶 M。单甲胺是该反应的竞争性抑制剂（Ki = 4.5 mM）。 DMA-MT 的表观分子量为 100 kDa，并且发现该酶是二聚体，由相同的 50 kDa 亚基组成。通过 HPLC 分析计算出类咕啉含量为 0.9 +/- 0.1 mol B12/mol 全酶。分离的甲基转移酶没有活性，但可以通过还原重新激活。激活需要甲基转移酶激活蛋白、ATP 和二甲胺的存在。与这些化合物一起孵育导致咕啉辅基甲基化。
Dimethylamine:5-hydroxybenzimidazolylcobamide methyltransferase (DMA-MT) was purified from cells of Methanosarcina barkeri Fusaro grown on trimethylamine. In the presence of methylcobalamine:coenzyme M methyltransferase isoenzyme II [MT2(II)] the enzyme quite specifically catalyzed the stoichiometric conversion of dimethylamine (apparent Km = 0.45 mM) and 2-mercaptoethane-sulfonate (coenzyme M) to monomethylamine and methyl-coenzyme M. Monomethylamine was a competitive inhibitor of the reaction (Ki = 4.5 mM). The apparent molecular mass of DMA-MT was 100 kDa and the enzyme was found to be a dimer, composed of identical 50-kDa subunits. A corrinoid content of 0.9 +/- 0.1 mol B12/mol holoenzyme was calculated from HPLC analysis. The as-isolated methyltransferase was inactive, but it could be reductively reactivated. Activation required the presence of methyltransferase-activating protein, ATP and dimethylamine. Incubation with these compounds resulted in the methylation of the corrinoid prosthetic group.