In microbial corrinoid-dependent methyltransferase systems, adventitious Co(I)-corrinoid oxidation halts catalysis and necessitates repair by ATP-dependent reductive activases. RamA, an activase with a C-terminal ferredoxin domain with two [4Fe-4S] clusters from methanogenic archaea has been far less studied than the bacterial activases bearing an N-terminal ferredoxin domain with one [2Fe-2S] cluster. These differences suggest RamA might prove to have other distinctive characteristics. Here, we examine RamA kinetics and the stoichiometry of the corrinoid protein:RamA complex. Like bacterial activases, K⁺ stimulates RamA. Potassium stimulation had been questioned due to differences in the primary structure of bacterial and methanogen activases. Unlike one bacterial activase, ATP is not inhibitory allowing the first determination of apparent kinetic parameters for any corrinoid activase. Unlike bacterial activases, a single RamA monomer complexes a single corrinoid protein monomer. Alanine replacement of a RamA serine residue corresponding to the serine of one bacterial activase which ligates the corrinoid cobalt during complex formation led to only moderate changes in the kinetics of RamA. These results reveal new differences in the two types of corrinoid activases, and provide direct evidence for the proposal that corrinoid activases act as catalytic monomers, unlike other enzymes that couple ATP hydrolysis to difficult reductions.

中文翻译：

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RamA的动力学和底物复合物表征，RamA是一种来自巴氏甲烷八叠球菌的类Corrinoid蛋白还原活化酶。

在微生物类corrinoid依赖的甲基转移酶系统中，不定Co（I）-corrinoid氧化可停止催化作用，并需要通过ATP依赖的还原活化酶进行修复。RamA是一种具有C端铁氧还蛋白结构域和两个产甲烷菌的[4Fe-4S]簇的活化酶，其活性远低于带有一个N端铁氧还蛋白结构域和一个[2Fe-2S]簇的细菌激活酶。这些差异表明RamA可能具有其他独特的特征。在这里，我们检查了RamA动力学和类固醇蛋白：RamA复合物的化学计量。像细菌激活酶一样，K ⁺刺激RamA。由于细菌和产甲烷激活酶的一级结构存在差异，人们对钾的刺激提出了质疑。与一种细菌激活酶不同，ATP不具有抑制性，因此可以首先确定任何类固醇激活酶的表观动力学参数。与细菌激活酶不同，单一的RamA单体会与单一的类皮质醇蛋白单体复合。丙氨酸替换对应于一​​种细菌活化酶丝氨酸的RamA丝氨酸残基的丝氨酸在复合物形成过程中连接到类蛋白钴的丝氨酸，仅导致RamA动力学的适度变化。这些结果揭示了两种类型的类Corrinoid活化酶之间的新差异，并为该类Corrinoid活化酶充当催化单体的提议提供了直接证据，这不同于将ATP水解偶联至难以还原的其他酶。