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dc.contributor.advisor Rajagopalan, K. V. en_US
dc.contributor.author Qiu, James en_US
dc.date.accessioned 2009-08-27T18:39:02Z
dc.date.available 2011-07-26T04:30:04Z
dc.date.issued 2009 en_US
dc.identifier.uri http://hdl.handle.net/10161/1332
dc.description Dissertation en_US
dc.description.abstract <p>Sulfite oxidase, a metabolically important enzyme, catalyzes the physiologically critical conversion of sulfite to sulfate in the terminal step of the degradation of sulfur containing compounds. The enzyme has been the focus for much research since its discovery in the 1950's. A central question to understanding the mechanism of molybdoenzymes such as sulfite oxidase and nitrate reductase concerns the roles of active site residues and the coordination chemistry of the Mo atom in the structure and function of the enzyme. The goal of this work was directed towards the characterization and determination of the structures of active site variants of sulfite oxidase using a spectroscopic, kinetic, and protein crystallographic approach.</p><p>Earlier studies have identified a single, highly conserved cysteine residue as the donor of a covalent bond from the protein to molybdenum in sulfite oxidase and nitrate reductase. The C185S and C185A variants of chicken sulfite oxidase exhibited severely attenuated activity. Crystallographic and spectroscopic analysis of both variants revealed a change in the metal coordination, from a dioxo to a trioxo form of Mo. </p><p>Assimilatory nitrate reductase is a member of the sulfite oxidase family of molybdopterin enzymes. The crystal structure of the Mo domain of the enzyme from Pichia angusta revealed high structural homology in the active sites of nitrate reductase and sulfite oxidase. Both enzymes utilize the same form of the molybdenum cofactor and have three out of five residues conserved at the active site. Substitution of two active site residues in sulfite oxidase alters the substrate affinity of chicken SO from sulfite to nitrate, resulting in an increase of nitrate reductase activity over wild-type sulfite oxidase. Additionally we identified an additional amino acid position in sulfite oxidase that corresponds to a non-conserved position in NR that further increased NR activity. Finally, these nitrate reductase variants of sulfite oxidase were crystallized and the structures solved. This represents the first example of the transmutation of a molybdenum enzyme to change activity and substrate affinity to those of a homologous enzyme.</p> en_US
dc.format.extent 26510307 bytes
dc.format.mimetype application/pdf
dc.language.iso en_US
dc.subject Chemistry, Biochemistry en_US
dc.subject Crystallography en_US
dc.subject Enzymology en_US
dc.subject Molybdenum en_US
dc.subject Nitrate Reductase en_US
dc.subject Structure en_US
dc.subject Function en_US
dc.subject Sulfite Oxidase en_US
dc.title Structure-Function Studies in Sulfite Oxidase with Altered Active Sites en_US
dc.type Dissertation en_US
dc.department Biochemistry en_US
duke.embargo.months 12 en_US
dc.date.accessible 2010-05-18T05:00:24Z

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