Perturbations to the active site of phosphotriesterase.
Perturbations to the active site of phosphotriesterase.
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DOI：
10.1021/bi962099l
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发表时间：
1997-02
期刊：
影响因子：
2.9
通讯作者：
J. Kuo;M. Chae;F. Raushel
中科院分区：
文献类型：
--
作者：
J. Kuo;M. Chae;F. Raushel
磷酸三酯酶催化有机磷神经毒剂的水解。四个氨基酸残基，位于活性位点口袋内，突变的努力，以确定这些基团在这种酶的结构和功能中发挥的作用。色氨酸-131位于双核金属中心的入口处，吲哚环的位置表明它可以为芳香族离去基团与优化底物的相互作用提供疏水位点。W131 F突变体显示出与野生型酶基本相同的对氧磷水解的催化常数。然而，W131 A突变体的Km值升高了6倍，这与该残基在底物结合中的作用一致。天冬氨酸-253与His-230形成氢键，而His-230又直接连接到更多溶剂暴露的金属离子上。D253 N突变体具有与野生型酶几乎相同的催化常数，而D253 A突变体的活性降低了500倍。这些结果与模型一致，其中需要该残基定向His-230的咪唑侧链以与双核金属中心进行适当的相互作用。天冬氨酸-301是更深埋的金属离子的主要配体。该残基突变为组氨酸、天冬酰胺、丙氨酸和半胱氨酸分别使催化活性降低2.6 x 10（4）、2.7 x 10（3）、5.6 x 10（2）和1.5 x 10（2）倍。这些结果表明，直接金属配体的改变，即使与残基，可以强烈地协调二价阳离子，导致严重破坏的活性位点的正常功能。在野生型酶中，Lys-169的侧链被氨甲酰化，并且还充当两个二价阳离子之间的桥。在该残基突变为丙氨酸、谷氨酸、精氨酸或甲硫氨酸后，催化活性显著损失。活性的损失可以在短链羧酸的测定混合物中包含后部分恢复。在100 mM丙酸存在下，观察到K169 A突变体的k（cat）增加了25倍。
Phosphotriesterase catalyzes the hydrolysis of organophosphate nerve agents. Four amino acid residues, located within the active site pocket, were mutated in an effort to ascertain the roles that these groups play in the structure and function of this enzyme. Tryptophan-131 is located at the entrance to the binuclear metal center, and the indole ring is positioned to suggest that it could provide a hydrophobic site for interaction of the aromatic leaving group with optimized substrates. The W131F mutant displays catalytic constants for the hydrolysis of paraoxon that are essentially the same as those of the wild type enzyme. However, the Km value for the W131A mutant is elevated by a factor of 6, consistent with a role for this residue in substrate binding. Aspartate-253 is hydrogen bonded to His-230 which, in turn, is directly ligated to the more solvent-exposed metal ion. The D253N mutant possesses catalytic constants that are virtually the same as those of the wild type enzyme, while the D253A mutant is reduced in activity by 500-fold. These results are consistent with a model where this residue is required to orientate the imidazole side chain of His-230 for proper interaction with the binuclear metal center. Aspartate-301 is a primary ligand to the more buried metal ion. Mutation of this residue to histidine, asparagine, alanine, and cysteine reduces the catalytic activity by factors of 2.6 x 10(4), 2.7 x 10(3), 5.6 x 10(2), and 1.5 x 10(2), respectively. These results indicate that alterations to the direct metal ligands, even with residues that can strongly coordinate divalent cations, cause a severe disruption to the proper functioning of the active site. In the wild type enzyme, the side chain of Lys-169 is carbamylated and also acts as a bridge between the two divalent cations. Significant losses in catalytic activity are obtained upon mutation of this residue to either alanine, glutamate, arginine, or methionine. The loss in activity can partially be restored upon inclusion in the assay mixture of short-chain carboxylic acids. A 25-fold enhancement in k(cat) is observed for the K169A mutant in the presence of 100 mM propionic acid.