Contribution of the active-site metal cation to the catalytic activity and to the conformational stability of phosphotriesterase:: temperature- and pH-dependence

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.