Crystal structure of 4-oxalocrotonate tautomerase inactivated by 2-oxo-3-pentynoate at 2.4 Å resolution:: Analysis and implications for the mechanism of inactivation and catalysis
Crystal structure of 4-oxalocrotonate tautomerase inactivated by 2-oxo-3-pentynoate at 2.4 Å resolution:: Analysis and implications for the mechanism of inactivation and catalysis
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DOI：
10.1021/bi981607j
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发表时间：
1998-10-20
期刊：
影响因子：
2.9
通讯作者：
Hackert, ML
中科院分区：
文献类型：
--
作者：
Taylor, AB;Czerwinski, RM;Hackert, ML
被活性位点定向不可逆抑制剂 2-oxo-3-pentynoate (2-OF) 灭活的 4-草酰巴豆酸互变异构酶 (4-OT) 的晶体结构已确定为 2.4 埃分辨率。通过所得 2-oxo-3-pentenoate 加合物对 Pro-1 进行共价修饰的酶的结构几乎与游离酶的结构重叠，并证实活性位点位于 Pro-1 周围的疏水区域。这两种结构都可以描述为二聚体的三聚体，其中每个二聚体由四链β-折叠组成，一侧有两个反平行的α-螺旋。对结构的检查还揭示了加合物和活性位点中的两个残基之间的非共价相互作用。来自相邻二聚体的Arg-39”的胍侧链的ε和eta氮分别与加合物的C-2羰基氧和一个C-1羧酸根氧相互作用，而来自与修饰的Pro-1相同二聚体的Arg-61'侧链与二齿中的C-1羧酸根相互作用活性位点区域内的两个有序水分子之一提供了与加合物的 2-氧代基团的额外相互作用，这些相互作用与 2-oxo-3-butynoate 是比 2-OF 更有效的 4-草酰巴豆酸互变异构酶抑制剂的观察结果相结合，表明 Arg-39” 和有序水分子极化了 2-OF 的羰基，从而促进了 Pro-1 和 Pro-1 之间的迈克尔反应。乙炔化合物。根据晶体结构，提出了酶催化反应的机理。
The crystal structure of 4-oxalocrotonate tautomerase (4-OT) inactivated by the active site-directed irreversible inhibitor 2-oxo-3-pentynoate (2-OF) has been determined to 2.4 Angstrom resolution. The structure of the enzyme covalently modified at Pro-1 by the resulting 2-oxo-3-pentenoate adduct is nearly superimposable on that of the free enzyme and confirms that the active site is located in a hydrophobic region surrounding Pro-1. Both structures can be described as a trimer of dimers where each dimer consists of a four-stranded beta-sheet with two antiparallel alpha-helices on one side. Examination of the structure also reveals noncovalent interactions between the adduct and two residues in the active site, The epsilon and eta nitrogens of the guanidinium side chain of Arg-39 " from a neighboring dimer interact respectively with the C-2 carbonyl oxygen and one C-1 carboxylate oxygen of the adduct while the side chain of Arg-61' from the same dimer as the modified Pro-1 interacts with the C-1 carboxylate group in a bidentate fashion. An additional interaction to the 2-oxo group of the adduct is provided by one of the two ordered water molecules within the active site region. These interactions coupled with the observation that 2-oxo-3-butynoate is a more potent irreversible inhibitor of 4-oxalocrotonate tautomerase than is 2-OF suggest that Arg-39 " and the ordered water molecule polarize the carbonyl group of 2-OF which facilitates a Michael reaction between Pro-1 and the acetylene compound. On the basis of the crystal structure, a mechanism for the enzyme-catalyzed reaction is proposed.