Goulding C W, Postigo D, Matthews R G
Biophysics Research Division, The University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
Biochemistry. 1997 Jul 1;36(26):8082-91. doi: 10.1021/bi9705164.
Methionine synthase (MetH) catalyzes the transfer of a methyl group from bound methylcobalamin to homocysteine, yielding enzyme-bound cob(I)alamin and methionine. The cofactor is then remethylated by methyltetrahydrofolate. We now demonstrate that MetH is able to catalyze methylation of free cob(I)alamin with methyltetrahydrofolate. MetH had previously been shown to catalyze methylation of homocysteine with free methylcobalamin as the methyl donor, in a reaction that is first-order in added methylcobalamin, and we have confirmed this observation using homogenous enzyme. A truncated polypeptide lacking the cobalamin-binding region of the holoenzyme, MetH(2-649), was overexpressed and purified to homogeneity. MetH(2-649) catalyzes the methylation of free cob(I)alamin by methyltetrahydrofolate and the methylation of homocysteine by free methylcobalamin. Furthermore, a protein comprising residues 2-353 of the holoenzyme has now been overexpressed and purified to homogeneity, and this protein catalyzes methyl transfer from free methylcobalamin to homocysteine but not from methyltetrahydrofolate to free cob(I)alamin. The mutations Cys310Ala and Cys311Ala in MetH(2-649) completely abolish methyl transfer from exogenous methylcobalamin to homocysteine but do not affect methyl transfer from methyltetrahydrofolate to exogenous cob(I)alamin, consistent with a modular construction for MetH. We infer that MetH is a modular protein comprising four separate regions: a homocysteine binding region (residues 2-353), a methyltetrahydrofolate binding region (residues 354-649), a region responsible for binding the cobalamin prosthetic group (residues 650-896), and an AdoMet-binding domain (residues 897-1227).
甲硫氨酸合酶（MetH）催化结合态甲基钴胺素上的甲基转移至同型半胱氨酸，生成酶结合态钴胺素（I）和甲硫氨酸。然后，辅因子由甲基四氢叶酸重新甲基化。我们现在证明，MetH能够催化游离钴胺素（I）与甲基四氢叶酸的甲基化反应。此前已证明MetH能够以游离甲基钴胺素作为甲基供体催化同型半胱氨酸的甲基化反应，该反应对添加的甲基钴胺素呈一级反应，我们使用均一酶证实了这一观察结果。一种缺失全酶钴胺素结合区域的截短多肽MetH(2 - 649)被过量表达并纯化至均一状态。MetH(2 - 649)催化游离钴胺素（I）与甲基四氢叶酸的甲基化反应以及游离甲基钴胺素与同型半胱氨酸的甲基化反应。此外，一种包含全酶2 - 353位残基的蛋白质现已被过量表达并纯化至均一状态，该蛋白质催化游离甲基钴胺素向同型半胱氨酸的甲基转移，但不催化甲基四氢叶酸向游离钴胺素（I）的甲基转移。MetH(2 - 649)中的Cys310Ala和Cys311Ala突变完全消除了外源甲基钴胺素向同型半胱氨酸的甲基转移，但不影响甲基四氢叶酸向外源钴胺素（I）的甲基转移，这与MetH的模块化结构一致。我们推断MetH是一种模块化蛋白质，由四个独立区域组成：同型半胱氨酸结合区域（2 - 353位残基）、甲基四氢叶酸结合区域（354 - 649位残基）、负责结合钴胺素辅基的区域（650 - 896位残基）和一个腺苷甲硫氨酸结合结构域（897 - 1227位残基）。
