Guilhaudis L, Simorre J P, Blackledge M, Marion D, Gans P, Neuburger M, Douce R
Laboratoire de Résonance Magnétique Nucléaire, Institut de Biologie Structurale CEA-CNRS "Jean-Pierre Ebel", 41 rue Jules Horowitz, 38027 Grenoble CEDEX 1, France.
Biochemistry. 2000 Apr 18;39(15):4259-66. doi: 10.1021/bi992674w.
The lipoate containing H-protein plays a pivotal role in the catalytic cycle of the glycine decarboxylase complex (GDC), undergoing reducing methylamination, methylene transfer, and oxidation. The transfer of the CH(2) group is catalyzed by the T-protein, which forms a 1:1 complex with the methylamine-loaded H-protein (Hmet). The methylamine group is then deaminated and transferred to the tetrahydrofolate-polyglutamate (H(4)FGlu(n)) cofactor of T-protein, forming methylenetetrahydrofolate-polyglutamate. The methylamine group is buried inside the protein structure and highly stable. Experimental data show that the H(4)FGlu(n) alone does not induce transfer of the methylene group, and molecular modeling also indicates that the reaction cannot take place without significant structural perturbations of the H-protein. We have, therefore, investigated the effect of the presence of the T-protein on the stability of Hmet. Addition of T-protein without H(4)FGlu(n) greatly increases the rate of the unloading reaction of Hmet, reducing the activation energy by about 20 kcal mol(-1). Differences of the (1)H and (15)N chemical shifts of the H-protein in its isolated form and in the complex with the T-protein show that the interaction surface for the H-protein is localized on one side of the cleft where the lipoate arm is positioned. This suggests that the role of the T-protein is not only to locate the tetrahydrofolate cofactor in a position favorable for a nucleophilic attack on the methylene carbon but also to destabilize the H-protein in order to facilitate the unlocking of the arm and initiate the reaction.
含硫辛酸的H蛋白在甘氨酸脱羧酶复合体（GDC）的催化循环中起关键作用，经历还原甲基化、亚甲基转移和氧化过程。亚甲基的转移由T蛋白催化，T蛋白与负载甲胺的H蛋白（Hmet）形成1:1复合物。然后甲胺基团脱氨基并转移到T蛋白的四氢叶酸 - 多聚谷氨酸（H4FGlu(n)）辅因子上，形成亚甲基四氢叶酸 - 多聚谷氨酸。甲胺基团埋藏在蛋白质结构内部且高度稳定。实验数据表明，单独的H4FGlu(n)不会诱导亚甲基的转移，分子建模也表明，若无H蛋白的显著结构扰动，反应无法发生。因此，我们研究了T蛋白的存在对Hmet稳定性的影响。在没有H4FGlu(n)的情况下添加T蛋白会大大增加Hmet的卸载反应速率，使活化能降低约20千卡·摩尔-1。H蛋白以分离形式及其与T蛋白复合物形式的1H和15N化学位移差异表明，H蛋白的相互作用表面位于硫辛酸臂所在裂隙的一侧。这表明T蛋白的作用不仅是将四氢叶酸辅因子定位在有利于对亚甲基碳进行亲核攻击的位置，还在于使H蛋白不稳定，以便促进臂的解锁并启动反应。
