Contribution of the active-site metal cation to the catalytic activity and to the conformational stability of phosphotriesterase:: temperature- and pH-dependence
Contribution of the active-site metal cation to the catalytic activity and to the conformational stability of phosphotriesterase:: temperature- and pH-dependence
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DOI：
10.1042/bj20031861
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发表时间：
2004-06-15
影响因子：
4.1
通讯作者：
Masson, P
中科院分区：
文献类型：
--
作者：
Rochu, D;Viguié, N;Masson, P
磷酸三酯酶（PTE）可以解毒神经毒剂和有机磷农药。PTE活性中心的两个锌阳离子可以被其他过渡金属阳离子取代而不损失活性。此外，金属取代的pte表现出不同的催化性能。提高PTE的热稳定性是制备高效PTE突变体的前提。通过等电聚焦、毛细管电泳和稳态动力学分析，确定了活性位点阳离子Zn2+、Co2+和Cd2+对PTE同工型催化活性和构象稳定性的贡献。三种同工异构体的pI值分别为7.2、7.5和7.1，且电泳滴定曲线不重叠。整体结构的改变，导致功能性质的变化，被发现与结合阳离子的性质有关：离子半径和离子电负性分别与k -m和k（cat）相关。此外，各亚型的ph依赖性活性谱也不同。温度相关的活性谱显示，在温度小于或等于35°c时最大活性，随后是45-48°c附近的激活阶段，然后在60°c时完成失活。pte的热变性分析证明了激活相是由一个瞬态中间体引起的。最后，在pH为8 ~ 9.4时，不同pte的热稳定性随pH的降低而提高，金属阳离子调制稳定性（Zn2+-、Co2+-和Cd2+- pte的t值分别为60.5 ~ 67℃、58 ~ 64℃和53 ~ 64℃）不同。证明了PTE的最佳活性（Co2+ PTE）和最大稳定性（Zn2+-PTE）的要求。
Phosphotriesterase (PTE) detoxifies nerve agents and organophosphate pesticides. The two zinc cations of the PTE active centre can be substituted by other transition metal cations without loss of activity. Furthermore, metal-substituted PTEs display differences in catalytic properties. A prerequisite for engineering highly efficient mutants of PTE is to improve their thermostability. Isoelectric focusing, capillary electrophoresis and steady-state kinetics analysis were used to determine the contribution of the active-site cations Zn2+, Co2+ or Cd2+ to both the catalytic activity and the conformational stability of the corresponding PTE isoforms. The three isoforms have different pI values (7.2, 7.5 and 7.1) and showed non-superimposable electrophoretic titration curves. The overall structural alterations, causing changes in functional properties, were found to be related to the nature of the bound cation: ionic radius and ion electronegativity correlate with K-m and k(cat) respectively. In addition, the pH-dependent activity profiles of isoforms were different. The temperature-dependent profiles of activity showed maximum activity at T less than or equal to 35 degreesC, followed by an activation phase near 45-48 degreesC and then inactivation which was completed at 60 degreesC. Analysis of thermal denaturation of the PTEs provided evidence that the activation phase resulted from a transient intermediate. Finally, at the optimum activity between pH 8 and 9.4, the thermostability of the different PTEs increased as the pH decreased, and the metal cation modulated stability (Zn2+-, Co2+- and Cd2+-PTE showed different T. values of 60.5-67 degreesC, 58-64 degreesC and 53-64 degreesC respectively). Requirements for optimum activity of PTE (displayed by Co2+ PTE) and maximum stability (displayed by Zn2+-PTE) were demonstrated.