The purification and characterization of the phosphotriesterase from Pseudomonas diminuta

Abstract

The phosphotriesterase from the soil bacterium Pseudomonas diminuta has been purified to homogeneity from two different expression systems using only three chromatographic steps. This represents the first total purification of any phosphotriester or phosphofluoridate hydrolyzing enzyme. The enzyme exists as a single subunit with a molecular weight of 39,000 and a Stoke's radius of 25 Å. There is no glycosylation of the baculovirus enzyme, but there is a single zinc atom at the active site that is required for enzymatic activity. Phosphotriesterase is capable of hydrolyzing a wide range of substrates at substantial rates in spite of the short 50 year history of phosphotriesters. The catalytic rate constant for paraoxon hydrolysis is 2100 s^-1. The chemical warfare agents sarin and soman are hydrolyzed at slower rates of 56 s^-1 and 5 s^-1 , respectively. The compound commonly used as a analog of these agents, diisopropylfluorophosphate, is hydrolyzed with a catalytic constant of 41 s^-1. The pH rate profile for paraoxon hydrolysis is characterized by the ionization of a single base with a pKa of 6.1 [plus or minus] 0.1 at 25 °C. The enthalpy of ionization for this base is 7.9 kcal/mol, and there is no shift in pK[a] in the presence of 20% DMF. These observations support the identity of the active site base as a histidyl residue. Furthermore, the chemical modification of phosphotriesterase resulted in inactivation only with the histidine modification reagents rose Bengal, methylene blue, and diethyl pyrocarbonate. There is complete protection against inactivation in the presence of diethyl p-fluorophenylphosphate. The pK[jnact] at 25 °C is 6.1 [plus or minus] 0.1, and the inactivation with diethyl pyrocarbonate resulted in an increase in absorbance characteristic to the modification of histidine residues at 245 nm. The expression of phosphotriesterase in fall army worms resulted in the production of about 11 enzymatic units per caterpillar after five days. While this amount is low in terms of output, this amount did provide a 280-fold increase in the LD[50] for topically applied paraoxon. This system, therefore, provides the first model for the induction of pesticide resistance in insects through the introduction of recombinant DNA.