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dc.contributor.advisorDeRose, Victoria J.en_US
dc.creatorSamples, Cynthia Reneeen_US
dc.date.accessioned2010-01-14T23:58:21Zen_US
dc.date.accessioned2010-01-16T02:23:01Z
dc.date.available2010-01-14T23:58:21Zen_US
dc.date.available2010-01-16T02:23:01Z
dc.date.created2005-12en_US
dc.date.issued2009-05-15en_US
dc.identifier.urihttp://hdl.handle.net/1969.1/ETD-TAMU-1010
dc.description.abstractPhosphotriesterase (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.en_US
dc.format.mediumelectronicen_US
dc.format.mimetypeapplication/pdfen_US
dc.language.isoen_USen_US
dc.subjectPTEen_US
dc.subjectphosphotriesteraseen_US
dc.subjectmanganeseen_US
dc.subjectEPRen_US
dc.subjectenzymesen_US
dc.subjectamidohydrolase superfamilyen_US
dc.subjectbinuclear active siteen_US
dc.titleInvestigation of the mechanism of phosphotriesterase: characterization of the binuclear metal active site by electron paramagnetic resonance spectroscopyen_US
dc.typeBooken
dc.typeThesisen
thesis.degree.departmentChemistryen_US
thesis.degree.disciplineChemistryen_US
thesis.degree.grantorTexas A&M Universityen_US
thesis.degree.nameDoctor of Philosophyen_US
thesis.degree.levelDoctoralen_US
dc.contributor.committeeMemberDunbar, Kimen_US
dc.contributor.committeeMemberFitzpatrick, Paulen_US
dc.contributor.committeeMemberRaushel, Frank M.en_US
dc.type.genreElectronic Dissertationen_US
dc.type.materialtexten_US
dc.format.digitalOriginborn digitalen_US


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