Cobalamin-dependent methionine synthase from Escherichia coli:: Involvement of zinc in homocysteine activation
Cobalamin-dependent methionine synthase from Escherichia coli:: Involvement of zinc in homocysteine activation
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DOI：
10.1021/bi971988l
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发表时间：
1997-12-16
期刊：
影响因子：
2.9
通讯作者：
Matthews, RG
中科院分区：
文献类型：
--
作者：
Goulding, CW;Matthews, RG
蛋氨酸合成酶（Methionine synthase, MetH）是一种具有至少四个不同区域的模块化蛋白质；氨基酸2-353包含一个负责结合和激活同型半胱氨酸的区域，氨基酸345-649被认为参与甲基四氢叶酸的结合和激活，氨基酸650-896负责结合假基甲基钴胺素，氨基酸897-1227参与结合腺苷蛋氨酸，并且是cob(II)alamin形式酶的还原激活所必需的。先前的研究表明，Cys310或Cys311突变为丙氨酸或丝氨酸导致所有可检测的催化活性丧失，这些突变蛋白保留了催化甲基从甲基四氢叶酸转移到外源cob(I)alamin的能力，但失去了将甲基从外源甲基钴胺转移到同型半胱氨酸的能力[Goulding， c.w, Postigo， D., and Matthews， R. G. (1997) Biochemistry 36, 8082-8091]。我们现在证明了甲基全酶和缺少钴胺素假基的截断甲基（2-649）蛋白含有0.9等量锌，而Cys310Ser和Cys311Ser突变蛋白含有少于0.05等量锌。将l -同型半胱氨酸添加到甲基（2-649）中会释放1等量的质子/mol蛋白质，而将l -同型半胱氨酸添加到Cys310Ser和Cys311Ser突变体截短的蛋白质中不会导致质子释放。Cys310Ala和Cys311Ala突变体甲基钴胺素全酶完全失去了将甲基从甲基钴胺素转移到同型半胱氨酸的能力，这表明锌是该反应所必需的。进一步的证据表明，锌是催化活性所必需的，在实验中，锌是从甲基中去除的（2-1227）。从甲基化的野生型全酶中去除锌，先用甲基甲乙硫基磺酸盐处理，然后用二硫苏糖醇和EDTA处理，导致蛋白质失去催化甲基从甲基四氢叶酸转移到同型半胱氨酸的能力。失锌全酶的重组导致0.4等量锌/mol蛋白质的掺入，部分恢复了蛋白质催化同型半胱氨酸甲基化的能力。
Methionine synthase (MetH) is a modular protein with at least four distinct regions; amino acids 2-353 comprise a region responsible for binding and activation of homocysteine, amino acids 345-649 are thought to be involved in the binding and activation of methyltetrahydrofolate, amino acids 650-896 are responsible for binding of the prosthetic group methylcobalamin, and amino acids 897-1227 are involved in binding adensylmethionine and are required for reductive activation of enzyme in the cob(II)alamin form. Previous studies have shown that mutations of Cys310 or Cys311 to either alanine or serine result in loss of all detectable catalytic activity, These mutant proteins retain the ability to catalyze methyl transfer from methyltetrahydrofolate to exogenous cob(I)alamin, but have lost the ability to transfer methyl groups from exogenous methylcobalamin to homocysteine [Goulding, C. W,, Postigo, D., and Matthews, R. G. (1997) Biochemistry 36, 8082-8091], We now demonstrate that both MetH holoenzyme and a truncated MetH(2-649) protein, which lacks a cobalamin prosthetic group, contain 0.9 equiv of zinc, while the Cys310Ser and Cys311Ser mutant proteins contain less than 0.05 equiv of zinc. Addition of L-homocysteine to MetH(2-649) is accompanied by release of 1 equiv of protons/mol of protein, while addition of L-homocysteine to the Cys310Ser and Cys311Ser mutant truncated proteins does not result in proton release. The Cys310Ala and Cys311Ala mutant methylcobalamin holoenzymes have completely lost the ability to transfer the methyl group from methylcobalamin to homocysteine, suggesting that zinc is required for this reaction. Further evidence that zinc is required for catalytic activity comes from experiments in which the zinc is removed from MetH(2-1227), Removal of zinc from methylated wild-type holoenzyme by treatment with methyl methanethiolsulfonate and then with dithiothreitol and EDTA results in loss of the ability of the protein to catalyze methyl transfer from methyltetrahydrofolate to homocysteine. Reconstitution of the zinc-depleted holoenzyme results in incorporation of 0.4 equiv of zinc/mol of protein and partial restoration of the ability of the protein to catalyze homocysteine methylation.