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首页> 外文期刊>Biochemistry >Synergistic, random sequential binding of substrates in cobalamin-independent methionine synthase
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Synergistic, random sequential binding of substrates in cobalamin-independent methionine synthase

机译：与钴胺素不相关的蛋氨酸合酶中底物的协同，随机顺序结合

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摘要

Cobalamin-independent methionine synthase (MetE) catalyzes the transfer of the N5-methyl group of methyltetrahydrofolate (CH3-H(4)folate) to the sulfur of homocysteine (Hcy) to form methionine and tetrahydrofolate (H(4)folate) as products. This reaction is thought to involve a direct methyl transfer from one substrate to the other, requiring the two substrates to interact in a ternary complex. The crystal structure of a MetE(.)CH(3)-H(4)folate binary complex shows that the methyl group is pointing away from the Hcy binding site and is quite distant from the position where the Sulfur of Hcy would be, raising the possibility that this binary complex is nonproductive. The CH3-H(4)folate must either rearrange or dissociate before methyl transfer can occur. Therefore, determining the order of substrate binding is of interest. We have used kinetic and equilibrium measurements in addition to isotope trapping experiments to elucidate the kinetic pathway of substrate binding in MetE. These studies demonstrate that both substrate binary complexes are chemically and kinetically competent for methyl transfer and suggest that the conformation observed in the crystal structure is indeed on-pathway. Additionally, the substrates are shown to bind synergistically, with each substrate binding 30-fold more tightly in the presence of the other. Methyl transfer has been determined to be slow compared to ternary complex formation and dissociation. Simulations indicate that nearly all of the enzyme is present as the ternary complex under physiological conditions.
机译：不依赖钴胺素的蛋氨酸合酶（MetE）催化将甲基四氢叶酸（CH3-H（4）叶酸）的N5-甲基转移至高半胱氨酸（Hcy）的硫中形成蛋氨酸和四氢叶酸（H（4）叶酸）产品。认为该反应涉及从一种底物到另一种底物的直接甲基转移，需要两种底物在三元络合物中相互作用。 MetE（。）CH（3）-H（4）叶酸二元配合物的晶体结构表明，甲基指向远离Hcy结合位点的位置，并且远离Hcy硫的位置，从而提高了这种二元复合物是非生产性的可能性。 CH3-H（4）叶酸必须重新排列或解离才能发生甲基转移。因此，确定底物结合的顺序是令人关注的。除同位素捕获实验外，我们还使用了动力学和平衡测量来阐明MetE中底物结合的动力学途径。这些研究表明，两种底物二元配合物在化学和动力学上均具有甲基转移的能力，并表明在晶体结构中观察到的构象确实在路上。另外，显示出底物协同结合，在存在另一底物的情况下，每种底物结合更紧密30倍。与三元复合物的形成和解离相比，甲基转移已被确定是缓慢的。模拟表明在生理条件下几乎所有的酶都以三元复合物形式存在。

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