Atomic resolution crystallography of a complex of triosephosphate isomerase with a reaction-intermediate analog: New insight in the proton transfer reaction mechanism
Atomic resolution crystallography of a complex of triosephosphate isomerase with a reaction-intermediate analog: New insight in the proton transfer reaction mechanism
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DOI：
10.1002/prot.22701
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发表时间：
2010-06-01
影响因子：
2.9
通讯作者：
Wierenga, Rik K.
中科院分区：
文献类型：
--
作者：
Alahuhta, Markus;Wierenga, Rik K.
酶通过精确定位底物和催化残基彼此之间的位置来实现其催化能力。原子分辨率晶体学是研究这些几何活性位特征的重要细节的极好工具。在这里，我们研究了磷酸丙糖异构酶（TIM）的反应机制，使用原子分辨率晶体学研究在0.82埃分辨率的利什曼醛TIM复杂的反应中间体类似物磷酸羟肟酸（PGH）。TIM的反应机制，如第一反应中间体的质子化状态和活性位点中的氢键相互作用的性质的剩余未解决的方面正在解决。PGH的异羟肟酸酯部分通过其N1-O 1部分的异常短的氢键与催化谷氨酸盐（Glu 167）的羧酸酯基团相互作用，例如，N1（PGH）-OE 2（Glu 167）的距离为2.69 +/-0.01埃，O 1（PGH）-OE 1（Glu 167）的距离为2.60 +/-0.01埃。结构比较表明，侧链的催化碱（Glu 167）可以移动在反应周期中的一个小腔，位于上面的异羟肟酸平面。结构分析表明PGH的异羟肟酸部分带负电荷。因此，结合的PGH模拟带负电荷的烯二醇中间体，其在通过催化谷氨酸从DHAP初始质子提取后立即形成。的TIM反应机制的背景下，目前的知识的新发现进行了讨论。
Enzymes achieve their catalytic proficiency by precisely positioning the substrate and catalytic residues with respect to each other. Atomic resolution crystallography is an excellent tool to study the important details of these geometric active-site features. Here, we have investigated the reaction mechanism of triosephosphate isomerase (TIM) using atomic resolution crystallographic studies at 0.82-angstrom resolution of leishmanial TIM complexed with the well-studied reaction-intermediate analog phosphoglycolohydroxamate (PGH). Remaining unresolved aspects of the reaction mechanism of TIM such as the protonation state of the first reaction intermediate and the properties of the hydrogen-bonding interactions in the active site are being addressed. The hydroxamate moiety of PGH interacts via unusually short hydrogen bonds of its N1-O1 moiety with the carboxylate group of the catalytic glutamate (Glu167), for example, the distance of N1(PGH)-OE2(Glu167) is 2.69 +/- 0.01 angstrom and the distance of O1(PGH)-OE1(Glu167) is 2.60 +/- 0.01 angstrom. Structural comparisons show that the side chain of the catalytic base (Glu167) can move during the reaction cycle in a small cavity, located above the hydroxamate plane. The structure analysis suggests that the hydroxamate moiety of PGH is negatively charged. Therefore, the bound PGH mimics the negatively charged enediolate intermediate, which is formed immediately after the initial proton abstraction from DHAP by the catalytic glutamate. The new findings are discussed in the context of the current knowledge of the TIM reaction mechanism.