The wild type bacterial Co 2+/Co 2+-phosphotriesterase shows a middle-range thermostability

Abstract

The phosphotriesterase (PTE) from Pseudomonas diminuta, a metalloenzyme that catalyses the hydrolysis of organophosphorus pesticides and nerve agents, has been described as a remarkably heat-stable protein [Grimsley et al., Biochemistry 36 (1997), 14366–14374]. Because substitution of the naturally occurring zinc ions by cobalt ions was found to enhance the enzyme catalytic activity, we investigated the thermal stability of the Co 2+/Co 2+-PTE. This study, carried out using capillary electrophoresis under optimised conditions in the pH range 9–10 compatible with optimal enzyme activity, provided evidence for irreversible denaturation according to the Lumry–Eyring model. A temperature-induced conformational transition ( T m≈58°C) and an early growing of aggregates were observed. Comparison of UV spectra with heat-induced inactivation data clearly demonstrated that the PTE state populated above T m was neither native nor active. Differential scanning calorimetry showed only an exothermic trace due to aggregation of the denatured protein at T=76°C. Accordingly, the temperature-induced denaturation process of the PTE could be described by a consecutive reaction model, including formation of an intermediate with enhanced activity at T≈45°C and an inactive unfolded state populated at T≈58°C, which leads to denatured aggregates. Thus, the wild type Co 2+/Co 2+-PTE displays a middle-range thermostability. Hence, for decontamination purposes under extreme Earth temperatures, wild type and engineered mutants of PTE substituted with other metal cations should be evaluated.