Three-dimensional structure of the zinc-containing phosphotriesterase with the bound substrate analog diethyl 4-methylbenzylphosphonate
Three-dimensional structure of the zinc-containing phosphotriesterase with the bound substrate analog diethyl 4-methylbenzylphosphonate
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DOI：
10.1021/bi960325l
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发表时间：
1996-05-14
期刊：
影响因子：
2.9
通讯作者：
Holden, HM
中科院分区：
文献类型：
--
作者：
Vanhooke, JL;Benning, MM;Holden, HM
来自 Pseudomonas diminuta 的磷酸三酯酶催化对氧磷和相关乙酰胆碱酯酶抑制剂的水解，其速率增强接近 10(12)。该酶需要双核金属中心才能发挥活性，并且分离后的每个亚基含有 2 当量的锌。在这里，我们描述了 Zn2+/Zn2+ 取代酶与底物类似物 4-甲基苄基膦酸二乙酯复合的三维结构。研究中使用的晶体属于空间群 C2，晶胞尺寸为 a = 129.6 埃，b = 91.4 埃，c = 69.4 埃，β = 91.9 度，并且不对称单元中有两个亚基。该模型通过最小二乘分析精炼至标称分辨率为 2.1 埃，所有测量的 X 射线数据的晶体学 R 因子为 15.4%。与之前报道的含镉酶的结构一样，桥连配体是氨甲酰化赖氨酸残基（Lys 169）和氢氧化物。锌离子之间的距离为 3.3 埃。埋藏较多的锌离子被 His 55、His 57、Lys 169、Asp 301 和桥接氢氧化物包围，呈三角双锥排列，如镉取代酶所述。然而，与更多溶剂暴露的镉离子观察到的八面体配位不同，第二个锌以四面体方式连接至 Lys 169、His 201、His 230，和桥接氢氧化物。 4-甲基苄基膦酸二乙酯占据双核金属中心附近的位点，底物类似物的磷酰氧位于距更多溶剂暴露的锌离子 3.5 埃处。抑制剂的芳香部分结合在相当疏水的口袋中，活性位点口袋的一个显着特征是抑制剂和蛋白质之间缺乏直接的静电相互作用，这很可能解释了磷酸三酯酶表现出的广泛底物特异性。抑制剂在活性位点内的位置表明水解反应的亲核试剂是金属结合的氢氧化物。
Phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of paraoxon and related acetylcholinesterase inhibitors with rate enhancements that approach 10(12). The enzyme requires a binuclear metal center for activity and as isolated contains 2 equiv of zinc per subunit. Here we describe the three-dimensional structure of the Zn2+/Zn2+-substituted enzyme complexed with the substrate analog diethyl 4-methylbenzylphosphonate. Crystals employed in the investigation belonged to the space group C2 with unit cell dimensions of a = 129.6 Angstrom, b = 91.4 Angstrom, c = 69.4 Angstrom, beta = 91.9 degrees, and two subunits in the asymmetric unit. The model was refined by least-squares analysis to a nominal resolution of 2.1 Angstrom, and a crystallographic R-factor of 15.4% for all measured X-ray data. As in the previously reported structure of the cadmium-containing enzyme, the bridging ligands are a carbamylated lysine residue (Lys 169) and a hydroxide. The zinc ions are separated by 3.3 Angstrom. The more buried zinc ion is surrounded by His 55, His 57, Lys 169, Asp 301, and the bridging hydroxide in a trigonal bipyramidal arrangement as described for the cadmium-substituted enzyme, Unlike the octahedral coordination observed for the more solvent-exposed cadmium ion, however, the second zinc is tetrahedrally ligated to Lys 169, His 201, His 230, and the bridging hydroxide. The diethyl 4-methylbenzylphosphonate occupies a site near the binuclear metal center with the phosphoryl oxygen of the substrate analog situated at 3.5 Angstrom from the more solvent-exposed zinc ion. The aromatic portion of the inhibitor binds in a fairly hydrophobic pocket, A striking feature of the active site pocket is the lack of direct electrostatic interactions between the inhibitor and the protein, This most likely explains the broad substrate specificity exhibited by phosphotriesterase. The position of the inhibitor within the active site suggests that the nucleophile for the hydrolysis reaction is the metal-bound hydroxide.