Browsing by Subject "phosphotriesterase"
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Item Improving Reactivity Against Target Organothiophosphates via Active-Site Directed Mutagenisis of a Bacterial Phosphotriesterase(2013-01-17) Githens, Tyler 1986-Phosphotriesters, also known as organophosphates (OP), represent a class of toxic compounds first synthesized in Germany. Enzymatic removal of harmful insecticides and breakdown products is a promising alternative to skimming or dredging. Wild type bacterial phosphotriesterase (PTE) was screened against 7 agricultural organophosphates: coumaphos, chlorpyrifos, fenitrothion, temephos, profenofos, pirimiphosmethyl and diazinon. The initial results laid the groundwork for a mutagenesis study to investigate the determining factors in enzyme reactivity. Coumaphos is hydrolyzed more efficiently than any other target by the wild type cobalt enzyme (kcat/Km = 2 x 10^7 M^-1s^-1). Coumaphos, fenitrothion and chlorpyrifos had the lowest Km values from the initial screen and were targets for steady state kinetic characterization of active site mutants. Site directed mutagenesis of binding sites was conducted and the most reactive point mutants, F132G, F132V and S308G, were used as backgrounds for subsequent mutation. Seven active site double mutants: F132G/S308G, F132G/S308T, F132V/S308G, F132V/S308T, F132G/I106T, F132V/I106T and G308/W309 were purified to homogeneity for kinetic characterization. The double mutant G308/F132V enhanced chlorpyrifos reactivity relative to the wild type enzyme. This enhancement of reactivity is proposed to result from conformational rearrangement following substrate bond hydrolysis.Item Investigation of the mechanism of phosphotriesterase: characterization of the binuclear metal active site by electron paramagnetic resonance spectroscopy(2009-05-15) Samples, Cynthia ReneePhosphotriesterase (PTE) from Pseudomonas diminuta is a zinc metalloenzyme found in soil bacteria capable of organophosphate hydrolysis at rates approaching the diffusion controlled limit. Interest in PTE for degradation of chemical warfare agents and disposal of pesticides supports the need to understand the mechanism by which it performs hydrolysis. For further mechanistic clarity, this work will provide direct confirmation of the solvent bridge identity and the protonated species resulting in loss of catalytic identity. Inhibitor and product binding to the metal center will also be addressed; as well as the evaluation of the catalytic activity of Fe(II)-substituted PTE. This work has determined that the Mn/Mn-PTE electron paramagnetic resonance (EPR) spectrum exhibits exchange coupling that is facilitated through a hydroxide bridge. Protonation of the bridging hydroxide results in the loss of the exchange coupling between the two divalent cations and the loss of catalytic activity. The reversible protonation of the bridging hydroxide has an apparent pKa of 7.3 based upon changes in the EPR spectrum of Mn/Mn-PTE with alterations in pH. The pH-rate profile for the hydrolysis of paraoxon by Mn/Mn-PTE shows the requirement of a single function group that must be unprotonated with a pKa of 7.1. The comparable pKa values are proposed to result from the protonation of the same ionizable species. The effects of inhibitor and product binding on the magnetic properties of the metal center and the hydroxyl bridge are investigated by accessing new EPR spectral features. This work concludes that the binding of inhibitor occurs at the metal center and results in an increase of non-bridged hydroxyl species. These results, in conjunction with kinetic and crystallographic data, suggest that substrate binding via the phosphoryl oxygen at the ?-metal weakens the hydroxyl bridge coordination to the ?-metal. This loss of coordination would increase the nucleophilic character of the bridge, and binding of the substrate to the metal center would result in a stronger nucleophile for hydrolysis. Lastly, Fe(II) binding and activation of apoenzyme is evaluated under anaerobic conditions. This work concludes Fe/Fe-PTE is not catalytically active, but can bind up to 2 equivalent Fe(II) ions per active site.Item Mechanism for the hydrolysis of organophosphates and investigations into the stereoselective hydrolysis of organophosphorus Esters by Phosphotriesterase.(Texas A&M University, 2006-04-12) Aubert, Sarah DwyerPhosphotriesterase (PTE) is a zinc metalloenzyme that catalyzes the hydrolysis of organophosphorus compounds. Metal ion roles during binding and catalysis are probed by comparing the kinetic properties of Zn/Zn, Cd/Cd, and Zn/Cd PTE with a variety of phosphate trisesters. The metal in the α-site of the binuclear metal center modulates the pKa values determined from pH-rate profiles. These results suggest that the α-metal is responsible for activating the nucleophilic hydroxide. In an effort to determine the function of the β-metal, the kinetic parameters for diethyl p-chlorophenyl thiophosphate are compared with diethyl p-chlorophenyl phosphate. The thiophosphate substrate is hydrolyzed 20 to 100-times faster than the phosphate substrate for Zn/Zn, Cd/Cd, and Zn/Cd PTE. When Cd2+ occupies the β-site, the inverse thio effect increases which suggests polarization by the β-metal on the phosphoryl oxygen or sulfur bond. The catalytic roles of Asp 233, His 254, and Asp 301 are determined by comparing the kinetic parameters of a series of alanine and asparagine mutations with paraoxon and diethyl p-chlorophenyl phosphate. The increased rate of hydrolysis for diethyl p-chlorophenyl phosphate with the mutants is consistent with the existence of a proton relay system from Asp 301 to His 254 to Asp 233. A detailed mechanism for the hydrolysis of organophosphates by PTE has been proposed. PTE hydrolyzes a number of chiral organophosphorus esters. The pKa of the leaving group phenol is altered for a series of chiral phosphate, phosphonate, and phosphinate esters. The stereoselectivity of wild-type, G60A, and I106G/F132G/H257Y PTE is enhanced as the pKa value of the leaving group phenol increases for phosphate, phosphonate, and phosphinate esters. In addition to improving the stereoselectivity of phosphotriesterase, mutations that affect the size of the active site of PTE are screened to identify a mutant enzyme that preferentially hydrolyzes the opposite isomer of wild-type PTE. The rate constants and stereoselectivity ratios for a number of active site mutants have been determined. H254Y/L303T PTE reverses the stereoselective preference of phosphonate and phosphinate substrates. The PTE stereoselectivity of O-methyl, O-phenyl acetylphenyl phosphate is reversed 970-fold by I106G/F132G/H257Y. A reversal mutant was resolved for phosphate, phosphonate, and phosphinate esters.