The wild type bacterial Co(2+)/Co(2+)-phosphotriesterase shows a middle-range thermostability.
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) approximately equal to 58 degrees 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 degrees 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 approximately equal to 45 degrees C and an inactive unfolded state populated at T approximately equal to 58 degrees 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.
Rochu
Daniel
D
Unité d'Enzymologie, Centre de Recherches du Service de Santé des Armées, P.O. Box 87, 38702 La Tronche Cedex, France. danielrochu@crssa.fr
Beaufet
Nadège
N
Renault
Frédérique
F
Viguié
Nathalie
N
Masson
Patrick
P
D043303:Aryldialkylphosphatase; D002021:Buffers; D002384:Catalysis; D002413:Cations, Divalent; D003035:Cobalt / Q000737:chemistry*; D019075:Electrophoresis, Capillary; D004795:Enzyme Stability; D004950:Esterases / Q000737:chemistry*; D005417:Flavobacterium / Q000201:enzymology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D017510:Protein Folding; D011549:Pseudomonas / Q000201:enzymology*
D002021:Buffers; D002413:Cations, Divalent; D003035:Cobalt; D004950:Esterases; D043303:Aryldialkylphosphatase