title: Activation of the binuclear metal center through formation of phosphotriesterase-inhibitor complexes.
abstract: Phosphotriesterase (PTE) from Pseudomonas diminuta is a binuclear metalloenzyme that catalyzes the hydrolysis of organophosphate nerve agents at rates approaching the diffusion-controlled limit. The proposed catalytic mechanism postulates the interaction of the substrate with the metal center and subsequent nucleophilic attack by the bridging hydroxide. X-band EPR spectroscopy was utilized to monitor the active site of Mn/Mn-substituted PTE upon addition of two inhibitors, diisopropyl methyl phosphonate and triethyl phosphate, and the product of hydrolysis, diethyl phosphate. The effects of inhibitor and product binding on the magnetic properties of the metal center and the hydroxyl bridge were evaluated by measuring changes in the features of the EPR spectra. The EPR spectra support the proposal that the binding of substrate analogues to the binuclear metal center diminishes the population of hydroxide-bridged species. These results, in conjunction with previously published kinetic and crystallographic data, suggest that substrate binding via the phosphoryl oxygen at the beta-metal weakens the coordination of the hydroxide bridge to the beta-metal. The weakened coordination to the beta-metal ion increases the nucleophilic character of the hydroxide and is coupled to the increase in the electrophilic character of the substrate.
authors.affiliation: Department of Chemistry, P.O. Box 30012, Texas A&M University, College Station, Texas 77842-3012, USA.
authors.forename: Cynthia R
authors.initials: CR
authors.lastname: Samples
authors.forename: Frank M
authors.initials: FM
authors.lastname: Raushel
authors.forename: Victoria J
authors.initials: VJ
authors.lastname: DeRose
mesh_terms: D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D007700:Kinetics; D008345:Manganese / Q000737:chemistry*; D010755:Organophosphates / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000378:metabolism; D044345:Phosphoric Triester Hydrolases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D011485:Protein Binding; D011549:Pseudomonas / Q000201:enzymology
chemical_list: D010755:Organophosphates; D009943:Organophosphorus Compounds; D008345:Manganese; C023477:diisopropyl methylphosphonate; C034789:diethyl phosphate; D044345:Phosphoric Triester Hydrolases; C009549:triethyl phosphate