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dc.contributor.advisorFitzpatrick, Paul F.
dc.creatorSobrado, Pablo
dc.date.accessioned2004-09-30T02:11:57Z
dc.date.available2004-09-30T02:11:57Z
dc.date.created2003-08
dc.date.issued2004-09-30
dc.identifier.urihttp://hdl.handle.net/1969.1/565
dc.description.abstractFlavocytochrome b2 catalyzes the oxidation of lactate to pyruvate. Primary deuterium and solvent kinetic isotope effects have been used to determine the relative timing of cleavage of the lactate OH and CH bonds by the wild type enzyme, a mutant protein lacking the heme domain, and the D282N enzyme. The DVmax and D(V/Klactate) values are both 3.0, 3.6 and 4.5 for the wild type enzyme, flavin domain and D282N enzymes, respectively. The D20Vmax values are 1.38, 1.18, and 0.98 for the wild type enzyme, the flavin domain, and the D282N enzyme; the respective D20(V/Klactate) values are 0.9, 0.44, and 1.0. The Dkred value is 5.4 for the wild type enzyme and 3.5 for the flavin domain, whereas the D2Okred is 1.0 for both enzymes. The V/Klactate value for the flavin domain increases 2-fold at moderate concentrations of glycerol. The data are consistent with the lactate hydroxyl proton not being in flight in the transition state for CH bond cleavage and there being an internal equilibrium prior to CH bond cleavage which is sensitive to solution conditions. Removal of the hydroxyl proton may occur in this pre-equilibrium. Tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to indoleacetamide, carbon dioxide and water. Sequence alignments identified this enzyme as a member of the L-amino acid oxidase family. The tyrosine and arginine residues in L-amino acid oxidase that bind the carboxylate of o-aminobenzoate are conserved and correspond to Tyr413 and Arg98 in tryptophan 2-monooxygenase. Mutation and characterization of the Y413A, Y413F, R98K and R98A enzymes indicate that these residues are in the active site and interact with the substrate. Deletion of the OH group of Tyr413 increases the Kd for the substrate and makes CH bond cleavage totally rate limiting. The pH V/Ktrp rate profile for the Tyr413 mutant enzymes shows that this residue must be protonated for activity. For both the R98A and R98K enzymes flavin reduction is rate limiting. The Vmax and V/Ktrp pH profiles indicate that the unprotonated form of the substrate is the active form for activity.en
dc.format.extent3229782 bytesen
dc.format.extent211637 bytesen
dc.format.mediumelectronicen
dc.format.mimetypeapplication/pdf
dc.format.mimetypetext/plain
dc.language.isoen_US
dc.publisherTexas A&M Universityen
dc.subjectFlavoenzymeen
dc.subjectdeuterium kinetic isotope effectsen
dc.subjectmutant enzymesen
dc.titleStudies of the chemical mechanisms of flavoenzymesen
dc.typeBooken
dc.typeThesisen
thesis.degree.departmentBiochemistry and Biophysicsen
thesis.degree.disciplineBiochemistryen
thesis.degree.grantorTexas A&M Universityen
thesis.degree.nameDoctor of Philosophyen
thesis.degree.levelDoctoralen
dc.contributor.committeeMemberHu, James C.
dc.contributor.committeeMemberJohnson, Arthur E.
dc.contributor.committeeMemberReinhart, Gregory D.
dc.type.genreElectronic Dissertationen
dc.type.materialtexten
dc.format.digitalOriginborn digitalen


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