Kolhouse J F, Utley C, Stabler S P, Allen R H
Department of Medicine, University of Colorado Health Sciences Center, Denver 80262.
J Biol Chem. 1991 Dec 5;266(34):23010-5.
Methionine synthase catalyzes the conversion of N5-methyltetrahydrofolate and homocysteine to tetrahydrofolate and methionine. Methylcobalamin (Me-Cbl) is tightly bound to methionine synthase and is required for enzymatic activity. When added to crude tissue homogenates, Me-Cbl stimulates methionine synthase but similar stimulation is observed with hydroxocobalamin, cyanocobalamin (CN-Cbl), and adenosyl-Cbl, although the mechanisms involved are unknown. We prepared human apomethionine synthase and studied its activation in the presence of [14C]CN-Cbl and [14CH3]Me-Cbl with concentrations of 2-mercaptoethanol ranging from 0.15 to 100 mM. We observed that the removal of the labeled upper axial ligands from CN-Cbl and Me-Cbl both paralleled the activation of human apomethionine synthase. Spectral studies employing CN-Cbl and Me-Cbl showed that both forms of Cbl must be converted to Cob(II)alamin before they can bind to human apomethionine synthase and convert it to its activated holoenzyme form. Studies with 14 different Cbl analogues with alterations in various portions of the corrin ring and the nucleotide showed that all of the analogues were able to fully activate human methionine synthase when they were reduced with 2-mercaptoethanol. Full activation occurred at lower concentrations of many of the Cbl analogues than occurred with Cbl itself. We conclude that Me-Cbl and other forms of Cob(III)alamin do not bind to human apomethionine synthase and that all must first be reduced to Cob(II)alamin before such binding can occur. The fact that human methionine synthase shows little absolute specificity for alterations in various portions of the Cbl molecule suggests that the potent inhibition of mammalian methionine synthase activity observed in vivo with various Cbl analogues is due to inhibition of intracellular Cbl transport or to inhibition of the enzymatic formation of Cob(II)alamin rather than to direct inhibition of mammalian methionine synthase itself.
甲硫氨酸合酶催化N5-甲基四氢叶酸和高半胱氨酸转化为四氢叶酸和甲硫氨酸。甲钴胺（Me-Cbl）与甲硫氨酸合酶紧密结合，是酶活性所必需的。当添加到粗组织匀浆中时，Me-Cbl可刺激甲硫氨酸合酶，但用羟钴胺、氰钴胺（CN-Cbl）和腺苷钴胺也观察到类似的刺激作用，尽管其中涉及的机制尚不清楚。我们制备了人脱辅基甲硫氨酸合酶，并研究了在[14C]CN-Cbl和[14CH3]Me-Cbl存在下，2-巯基乙醇浓度在0.15至100 mM范围内时其激活情况。我们观察到，从CN-Cbl和Me-Cbl上去除标记的上位轴向配体均与人类脱辅基甲硫氨酸合酶的激活平行。使用CN-Cbl和Me-Cbl的光谱研究表明，两种形式的钴胺素都必须先转化为钴（II）钴胺素，才能与人脱辅基甲硫氨酸合酶结合并将其转化为活化的全酶形式。对14种不同的钴胺素类似物进行研究，这些类似物在咕啉环和核苷酸的不同部分有所改变，结果表明，当用2-巯基乙醇还原时，所有类似物都能够完全激活人甲硫氨酸合酶。许多钴胺素类似物在比钴胺素本身更低的浓度下就能实现完全激活。我们得出结论，Me-Cbl和其他形式的钴（III）钴胺素不会与人脱辅基甲硫氨酸合酶结合，所有这些都必须先还原为钴（II）钴胺素才能发生这种结合。人甲硫氨酸合酶对钴胺素分子不同部分的改变几乎没有绝对特异性，这一事实表明，在体内观察到的各种钴胺素类似物对哺乳动物甲硫氨酸合酶活性的有效抑制是由于抑制了细胞内钴胺素的转运，或者是抑制了钴（II）钴胺素的酶促形成，而不是直接抑制哺乳动物甲硫氨酸合酶本身。
