Activation of methyltetrahydrofolate by cobalamin-independent methionine synthase
Activation of methyltetrahydrofolate by cobalamin-independent methionine synthase
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DOI：
10.1021/bi060052m
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发表时间：
2006-04-25
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：
Taurog, RE;Matthews, RG
钴胺非依赖性蛋氨酸合成酶（MetE）以甲基四氢叶酸(CH3-H（4)PteGlu(3)）的甘油三酯衍生物为甲基供体，同型半胱氨酸（Hcy）为甲基受体，催化从头合成蛋氨酸的最后一步。这个反应是具有挑战性的，因为在生理pH下，Hcy硫醇不是强亲核试剂，CH3-H(4)PteGlu(3)提供了一个非常差的离去基。我们的实验室先前已经确定Hcy与MetE活性部位的紧密结合的锌离子相连。这种相互作用通过降低其pK(a)来激活Hcy，从而使硫代酸盐稳定在中性ph。剩下的化学挑战是活化CH3-H(4)PteGlu(3)。CH3-H-4-PteGlu(3)的N5质子化会产生更好的离去基，但溶液中的pK(a)为5。我们利用CH3-H(4)PteGlu(3)吸收光谱的灵敏度来探测其与MetE结合时的质子化状态。比较游离和甲基结合CH3-H(4)PteGlu(3)的吸光度光谱表明，N5在二元配合物中没有质子化。快速反应研究显示CH(3)H(4)PteGlu(3)吸光度的变化与N5的质子化一致。这些吸光度变化显示出对Hcy和CH3-H(4)PteGlu(3)的饱和依赖，表明CH3-H(4)PteGlu(3)的质子化发生在三级配合物形成时，在甲基转移之前。此外，四氢叶酸（H-4-PteGlu(3)）产物似乎仍然与MetE结合，并且在过量的Hcy存在下，MetE()H(4)PteGlu(3)（）。Hcy混合三元配合物，其中H(4)PteGlu(3)被质子化。
Cobalamin-independent methionine synthase (MetE) catalyzes the final step of de novo methionine synthesis using the triglutamate derivative of methyltetrahydrofolate (CH3-H(4)PteGlu(3)) as methyl donor and homocysteine (Hcy) as methyl acceptor. This reaction is challenging because at physiological pH the Hcy thiol is not a strong nucleophile and CH3-H(4)PteGlu(3) provides a very poor leaving group. Our laboratory has previously established that Hcy is ligated to a tightly bound zinc ion in the MetE active site. This interaction activates Hcy by lowering its pK(a), such that the thiolate is stabilized at neutral pH. The remaining chemical challenge is the activation of CH3-H(4)PteGlu(3). Protonation of N5 of CH3-H-4-PteGlu(3) would produce a better leaving group, but occurs with a pK(a) of 5 in solution. We have taken advantage of the sensitivity of the CH3-H(4)PteGlu(3) absorption spectrum to probe its protonation state when bound to MetE. Comparison of free and MetE-bound CH3-H(4)PteGlu(3) absorbance spectra indicated that the N5 is not protonated in the binary complex. Rapid reaction studies have revealed changes in CH(3)H(4)PteGlu(3) absorbance that are consistent with protonation at N5. These absorbance changes show saturable dependence on both Hcy and CH3-H(4)PteGlu(3), indicating that protonation of CH3-H(4)PteGlu(3) occurs upon formation of the tertiary complex and prior to methyl transfer. Furthermore, the tetrahydrofolate (H-4-PteGlu(3)) product appears to remain bound to MetE, and in the presence of excess Hcy a MetE(.)H(4)PteGlu(3)(.) Hcy mixed ternary complex forms, in which H(4)PteGlu(3) is protonated.