HYDROLYSIS OF PHOSPHODIESTERS THROUGH TRANSFORMATION OF THE BACTERIALPHOSPHOTRIESTERASE

The phosphotriesterase from Pseudomonas diminuta catalyzes the hydroly sis of a wide array of phosphotriesters and related phosphonates, incl uding organophosphate pesticides and military nerve agents. It has now been shown that this enzyme can also catalyze the hy drolysis of phos phodiesters, albeit at a greatly reduced rate. However, the enzymatic hydrolysis of ethyl-4-nitrophenyl phosphate (compound I) by the wild-t ype enzyme was >10(8) times faster than the uncatalyzed reaction (k(ca t) = 0.06 s(-1) and K-m = 38 mM). Upon the addition of various alkylam ines to the reaction mixture, the k(cat)/K-m for the phosphodiester (c ompound I) increased up to 200-fold. Four mutant enzymes of the phosph otriesterase were constructed in a preliminary attempt to improve phos phodiester hydrolysis activity of the native enzyme. Met-317, which is thought to reside in close proximity to the pro S-ethoxy arm of the p araoxon substrate, was mutated to arginine, alanine, histidine, and ly sine, These mutant enzymes showed slight improvements in the catalytic hydrolysis of organophosphate diesters, The M317K mutant enzyme displ ayed the most improvement in catalytic activity (k(cat) = 0.34 s(-1) a nd K-m = 30 mM), The M317A mutant enzyme catalyzed the hydrolysis of t he phosphodiester (compound I) in the presence of alkylamines up to 20 0 times faster than the wildtype enzyme in the absence of added amines , The neutralization of the negative charge on the oxygen atom of the phosphodiester by the ammonium cation within the active site is though t to be responsible for the rate enhancement by these amines in the hy drolytic reaction. These results demonstrate that an active site optim ized for the hydrolysis of organophosphate triesters can be made to ca talyze the hydrolysis of organophosphate diesters.