Chae M Y, Omburo G A, Lindahl P A, Raushel F M
Department of Chemistry, Texas A&M University, College Station 77843.
Arch Biochem Biophys. 1995 Feb 1;316(2):765-72. doi: 10.1006/abbi.1995.1102.
Bacterial phosphotriesterase catalyzes the hydrolysis of organophosphate triesters. To be active, the enzyme requires that two divalent cations are bound. These metal ions are bound in close proximity to one another as a binuclear center. To characterize the structure and function of the binuclear metal binding sites, we have prepared the copper-substituted enzyme. The kinetic data indicate that this enzyme is essentially inactive toward the hydrolysis of phosphotriesters. The EPR signal arising from the copper-substituted enzyme is nearly axial, with g parallel = 2.24 and g perpendicular = 2.05 and shows at least seven superhyperfine transitions in the g perpendicular region with A perpendicular = 1.45 x 10(-3) cm-1. These splittings are consistent with the direct ligation of more than one nitrogen to the metal center. The average spin quantitation of copper-substituted enzymes are 0.6 spin/Cu, approximately half of that observed for noninteracting Cu2+ ions. The spin intensity increases to ca. 1 spin/Cu when samples are denatured with acid. The binding of metal ions to the designated alpha and beta sites is highly synergistic (i.e., the metal ions bind in pairs). Mixed metal complexes of the type Cu/X and X/Cu were prepared. When X is a diamagnetic ion (Zn2+ or Cd2+), the spin quantitation increases, but when X is the paramagnetic Co2+ ion, the spin quantitation decreases. This behavior indicates that the low spin intensities observed for copper-substituted phosphotriesterase arise from spin-coupling of the two adjacent Cu2+ ions. The addition of dithiothreitol, ascorbate, or dithionite to the copper-substituted phosphotriesterase results in nearly the complete loss of spin intensity. This indicates that the bound coppers can be reduced to the cuprous state.
细菌磷酸三酯酶催化有机磷酸三酯的水解反应。该酶要具有活性，需要结合两个二价阳离子。这些金属离子以双核中心的形式紧密相邻结合。为了表征双核金属结合位点的结构和功能，我们制备了铜取代酶。动力学数据表明，该酶对磷酸三酯的水解基本无活性。铜取代酶产生的电子顺磁共振（EPR）信号几乎是轴向的，g平行 = 2.24，g垂直 = 2.05，并且在g垂直区域显示至少七个超超精细跃迁，A垂直 = 1.45×10⁻³ cm⁻¹。这些分裂与不止一个氮直接连接到金属中心一致。铜取代酶的平均自旋定量为0.6自旋/Cu，约为非相互作用Cu²⁺离子观察值的一半。当样品用酸变性时，自旋强度增加到约1自旋/Cu。金属离子与指定的α和β位点的结合具有高度协同性（即金属离子成对结合）。制备了Cu/X和X/Cu类型的混合金属配合物。当X是抗磁性离子（Zn²⁺或Cd²⁺）时，自旋定量增加，但当X是顺磁性Co²⁺离子时，自旋定量降低。这种行为表明，铜取代磷酸三酯酶观察到的低自旋强度是由两个相邻Cu²⁺离子的自旋耦合引起的。向铜取代磷酸三酯酶中加入二硫苏糖醇、抗坏血酸盐或连二亚硫酸盐会导致自旋强度几乎完全丧失。这表明结合的铜可以还原为亚铜状态。
