Abstract
The phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of a wide array of phosphotriesters and related phosphonates, including organophosphate pesticides and military nerve agents. It has now been shown that this enzyme can also catalyze the hydrolysis of phosphodiesters, albeit at a greatly reduced rate. However, the enzymatic hydrolysis of ethyl-4-nitrophenyl phosphate (compound I) by the wild-type enzyme was >10(8) times faster than the uncatalyzed reaction (kcat = 0.06 s-1 and Km = 38 mM). Upon the addition of various alkylamines to the reaction mixture, the kcat/Km for the phosphodiester (compound I) increased up to 200-fold. Four mutant enzymes of the phosphotriesterase were constructed in a preliminary attempt to improve phosphodiester hydrolysis activity of the native enzyme. Met-317, which is thought to reside in close proximity to the pro-S-ethoxy arm of the paraoxon substrate, was mutated to arginine, alanine, histidine, and lysine. These mutant enzymes showed slight improvements in the catalytic hydrolysis of organophosphate diesters. The M317K mutant enzyme displayed the most improvement in catalytic activity (kcat = 0.34 s-1 and Km = 30 mM). The M317A mutant enzyme catalyzed the hydrolysis of the phosphodiester (compound I) in the presence of alkylamines up to 200 times faster than the wild-type 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 thought to be responsible for the rate enhancement by these amines in the hydrolytic reaction. These results demonstrate that an active site optimized for the hydrolysis of organophosphate triesters can be made to catalyze the hydrolysis of organophosphate diesters.
Highlights
The bacterial phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of a wide range of organophosphate nerve agents with high efficiency [1, 2]
The role of one of the two metal ions within the active site is thought to involve the activation of the hydrolytic water molecule, whereas the companion metal ion is most likely involved in the polarization of the phosphoryl oxygen bond of the substrate to increase the electrophilicity of the substrate for nucleophilic attack [16]
Enzymatic Hydrolysis of Ethyl-4-nitrophenyl Phosphate (Compound I)—Compound I was tested as a substrate for the cobalt-substituted bacterial phosphotriesterase
Summary
The bacterial phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of a wide range of organophosphate nerve agents with high efficiency [1, 2]. The active site of this enzyme contains a coupled binuclear metal center, which is absolutely essential for catalytic activity [1]. An array of model compounds that promote the catalytic hydrolysis of phosphodiester bonds has been designed and synthesized [17,18,19,20,21,22,23]. Many enzymes are known to catalyze the hydrolysis of the phosphodiester bond, with kcat values ranging from 10Ϫ2 to 103 sϪ1. Since the catalytic machinery required for the hydrolysis of phosphodiesters is apparently similar to the binuclear metal center found within the active site of phosphotriesterase, we anticipated that phosphotriesterase would possess an inherent ability to hydrolyze phosphodiester substrates. The active site of phosphotriesterase is filled predominantly with hydrophobic residues, and this site may not be suitable for the accommodation of the negatively charged phosphodiesters. The rate enhancement can be intensified by the incorporation of a positive charge within the active site, either through site-directed mutagenesis or the addition of alkylamines to the aqueous medium
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