3-DIMENSIONAL STRUCTURE OF THE BINUCLEAR METAL CENTER OF PHOSPHOTRIESTERASE
3-DIMENSIONAL STRUCTURE OF THE BINUCLEAR METAL CENTER OF PHOSPHOTRIESTERASE
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DOI：
10.1021/bi00025a002
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发表时间：
1995-06-27
期刊：
影响因子：
2.9
通讯作者：
HOLDEN, HM
中科院分区：
文献类型：
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作者：
BENNING, MM;KUO, JM;HOLDEN, HM
从假单胞菌中分离出来的磷酸三酯酶能够解毒广泛使用的农药，例如对氧磷和对硫磷以及各种哺乳动物乙酰胆碱酯酶抑制剂。该酶需要双核金属中心才能发挥活性。最近，脱辅基酶的三维结构得到解决（Benning 等人，1994）并显示其由 α/β 桶组成。在这里，我们描述了用镉重构的全酶的三维结构，通过分辨率为 2.0 埃的 X 射线晶体学分析确定。研究中使用的晶体属于空间群 C2，晶胞尺寸为 a = 129.5 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。酶的 apo 和空穴形式的三维结构存在显着差异，因此它们的 cr-碳位置重叠，均方根偏差为 3.4 埃。双核金属中心位于β-桶的C末端，与镉相距3.8埃。金属有两个桥连配体：水分子（或可能是氢氧根离子）和氨甲酰化赖氨酸残基（Lys 169）。埋藏较多的镉被His 55、His 57、Lys 169、Asp 301和呈三角双锥排列的桥水包围。第二种金属由 His 201、His 230、Lys 169、桥联水分子和两种其他溶剂配位成扭曲的八面体几何形状。
Phosphotriesterase, as isolated from Pseudomonas diminuta, is capable of detoxifying widely used pesticides such as paraoxon and parathion and various mammalian acetylcholinesterase inhibitors. The enzyme requires a binuclear metal center for activity. Recently, the three-dimensional structure of the apoenzyme was solved (Benning et al., 1994) and shown to consist of an alpha/beta-barrel. Here we describe the three-dimensional structure of the holoenzyme, reconstituted with cadmium, as determined by X-ray crystallographic analysis to 2.0-Angstrom resolution. Crystals employed in the investigation belonged to the space group C2 with unit cell dimensions of a = 129.5 Angstrom, b = 91.4 Angstrom, c = 69.4 Angstrom, beta = 91.9 degrees, and two subunits in the asymmetric unit. There are significant differences in the three-dimensional architecture of the apo and hole forms of the enzyme such that their cr-carbon positions superimpose with a root-mean-square deviation of 3.4 Angstrom. The binuclear metal center is located at the C-terminus of the beta-barrel with the cadmiums separated by 3.8 Angstrom. There are two bridging ligands to the metals: a water molecule (or possibly a hydroxide ion) and a carbamylated lysine residue (Lys 169). The more buried cadmium is surrounded by His 55, His 57, Lys 169, Asp 301, and the bridging water in a trigonal bipyramidal arrangement. The second metal is coordinated in a distorted octahedral geometry by His 201, His 230, Lys 169, the bridging water molecule, and two additional solvents.