Product-assisted Catalysis as the Basis of the Reaction Specificity of Threonine Synthase
Product-assisted Catalysis as the Basis of the Reaction Specificity of Threonine Synthase
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DOI：
10.1074/jbc.m110.186205
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发表时间：
2011-01-28
影响因子：
4.8
通讯作者：
Hayashi, Hideyuki
中科院分区：
文献类型：
--
作者：
Murakawa, Takeshi;Machida, Yasuhiro;Hayashi, Hideyuki
苏氨酸合酶 (TS) 是一种吡哆醛 5'-磷酸 (PLP) 依赖性酶，催化消除 O-磷酸-L-高丝氨酸 (OPHS) 中的 γ-磷酸基团，并随后在 C beta 处添加水以形成 L-苏氨酸。 TS的催化过程是PLP酶中最复杂的，如何控制TS中的基本步骤来进行选择性反应是一个有趣的问题。当在磷酸盐存在下将 L-乙烯基甘氨酸添加到嗜热栖热菌 HB8 TS 中时，形成 L-苏氨酸，其 k(cat) 和反应特异性与使用 OPHS 作为底物时的反应特异性相当。然而，在没有磷酸盐的情况下或当使用硫酸盐代替磷酸盐时，仅形成副反应产物-α-酮丁酸酯。对TS与L-苏氨酸反应中光谱变化的整体分析表明，与酸性更强的硫酸根离子相比，磷酸根离子降低了PLP-α-氨基巴豆酸醛亚胺（AC）的Cβ处加水和转醛化形成L-苏氨酸的过渡态的能级。 TS 与 AC 类似物复合的 X 射线晶体学分析给出了独特的电子密度，该电子密度归属于源自类似物 C beta 附近溶剂的磷酸根离子。这些结果表明，通过γ-消除从OPHS中释放的磷酸根离子充当AC的Cβ处加水的碱催化剂，从而提供了反应专一性的基础。磷酸根离子也被认为可以加速 C γ 处的质子化/去质子化
Threonine synthase (TS), which is a pyridoxal 5 '-phosphate (PLP)-dependent enzyme, catalyzes the elimination of the gamma-phosphate group from O-phospho-L-homoserine (OPHS) and the subsequent addition of water at C beta to form L-threonine. The catalytic course of TS is the most complex among the PLP enzymes, and it is an intriguing problem how the elementary steps are controlled in TS to carry out selective reactions. When L-vinylglycine was added to Thermus thermophilus HB8 TS in the presence of phosphate, L-threonine was formed with k(cat) and reaction specificity comparable with those when OPHS was used as the substrate. However, in the absence of phosphate or when sulfate was used in place of phosphate, only the side reaction product-alpha-ketobutyrate, was formed. Global analysis of the spectral changes in the reaction of TS with L-threonine showed that compared with the more acidic sulfate ion, the phosphate ion decreased the energy levels of the transition states of the addition of water at the C beta of the PLP-alpha-aminocrotonate aldimine (AC) and the transaldimination to form L-threonine. The x-ray crystallographic analysis of TS complexed with an analog for AC gave a distinct electron density assigned to the phosphate ion derived from the solvent near the C beta of the analog. These results indicated that the phosphate ion released from OPHS by gamma-elimination acts as the base catalyst for the addition of water at C beta of AC, thereby providing the basis of the reaction specificity. The phosphate ion is also considered to accelerate the protonation/deprotonation at C gamma