ABSTRACT: Phosphotriesterase (PTE) is a binuclear metalloenzyme that catalyzes the hydrolysis of organophosphates, including pesticides and chemical warfare agents, at rates approaching the diffusion controlled limit. The catalytic mechanism of this enzyme features a bridging solvent molecule that is proposed to initiate nucleophilic attack at the phosphorus center of the substrate. X-band EPR spectroscopy is utilized to investigate the active site of Mn/Mn-substituted PTE. Simulation of the dominant EPR spectrum from the coupled binuclear center of Mn/Mn-PTE requires slightly rhombic zero-field splitting parameters. Assuming that the signal arises from the S ) 2 manifold, an exchange coupling constant of J ) -2.7 ( 0.2 cm -1 (H ex ) -2JS 1 ‚S 2 ) is calculated. A kinetic pK a of 7.1 ( 0.1 associated with loss in activity at low pH indicates that a protonation event is responsible for inhibition of catalysis. Analysis of changes in the EPR spectrum as a function of pH provides a pK a of 7.3 ( 0.1 that is assigned as the protonation of the hydroxyl bridge. From the comparison of kinetic and spectral pK a values, it is concluded that the loss of catalytic activity at acidic pH results from the protonation of the hydroxide that bridges the binuclear metal center. Phosphotriesterase (PTE) 1 catalyzes the hydrolysis of a wide range of organophosphate esters, including agricultural pesticides and chemical warfare agents (1-3). The enzyme has been isolated from soil bacteria, but the natural substrate for PTE is not known. PTE is a member of the amidohydrolase superfamily, which also includes urease, dihydroorotase, and approximately 30 other enzymes of known specificity (4). The high-resolution X-ray crystal structure of Zn/Zn-PTE reveals that it is a homodimeric protein containing an active site with two divalent metal ions embedded within a ( /R) 8 -barrel motif (5). The R-metal ion is ligated by His-55, His-57, and Asp-301 while the -metal ion is coordinated to His-201 and His-230 as illustrated i
