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dc.contributor.advisorRobertus, Jon D.en
dc.creatorWagner, Wendi Suzanneen
dc.date.accessioned2008-08-28T22:31:30Zen
dc.date.available2008-08-28T22:31:30Zen
dc.date.issued2004en
dc.identifierb60817318en
dc.identifier.urihttp://hdl.handle.net/2152/2071en
dc.descriptiontexten
dc.description.abstract5, 10-Methylenetetrahydrofolate dehydrogenase (yMTD) is an NAD+ - dependent enzyme located in the cytosol of Saccharomyces cerevisiae. It catalyzes the conversion of 5, 10-methylenetetrahydrofolate to 5, 10- methenyltetrahydrofolate, which then spontaneously oxidizes to 10- formyltetrahydrofolate within the cell. yMTD is one of three yeast isozymes, the other two of which have additional cyclohydrolase and synthetase activities, and is thought to be responsible for flux in the oxidative direction. To date, yMTD is the only known monofunctional eukaryotic MTD. The catalytic mechanism of yMTD was explored with site-directed mutagenesis of active site residues Glu121, Cys150, and T151 and with X-ray crystallography. Results showed Glu121 to be essential for catalysis, with mutations E121A and E121Q eliminating all measurable activity. E121D, which retains the carboxylic acid of Glu121, changed the kcat/Km to 10.1% of wild-type and the Km of substrate five-fold higher than wild-type. Mutations C150A, C150S and T151V also abolished activities. T151A reduced kcat/Km to 28.1% of wild-type. Crystal structures of E121A, C150A, and T151A did not show any conformational change from wild-type, indicating that mutations did not affect protein folding. Results suggest a catalytic mechanism in which Glu121 binds the substrate near carbon C2 to orient it for the hydride transfer to NAD+ . Cys150 contributes to the electron-rich environment of the substrate as well. Thr151 binds NAD+ with a hydrogen bond to the carbonyl of the nicotinamide ring and may help to stabilize the transition state. Mutations of active site residues A50S, T57K, and Y98Q were made in the hope of restoring cyclohydrolase activity to yMTD. These efforts were unsuccessful, although the cyclohydrolase assay is too crude to measure small amounts of activity. Various folate analogs were also tried against wild-type yMTD and pteroic acid, 5-formyltetrahydrofolate, and methotrexate were identified as inhibitors. These inhibitors were used in crystal screens against wild-type, E121A, and C150A yMTD to grow crystals of yMTD with a substrate analog. Although crystals were formed, they only grew under wild-type conditions, had the same unit cell as apoenzyme, and did not show density for a substrate analog in the refined structure.
dc.format.mediumelectronicen
dc.language.isoengen
dc.rightsCopyright is held by the author. Presentation of this material on the Libraries' web site by University Libraries, The University of Texas at Austin was made possible under a limited license grant from the author who has retained all copyrights in the works.en
dc.subject.lcshSaccharomyces cerevisiaeen
dc.subject.lcshYeast fungi--Molecular aspectsen
dc.subject.lcshFungal molecular biologyen
dc.titleCatalytic mechanism of Saccharomyces cerevisiae NAD+-dependent 5,10-methylenetetrahydrofolate dehydrogenaseen
dc.description.departmentChemistry and Biochemistryen
dc.description.departmentBiochemistryen
dc.identifier.oclc68810358en
dc.identifier.proqst3145871en
dc.type.genreThesisen
thesis.degree.departmentBiochemistryen
thesis.degree.disciplineBiochemistryen
thesis.degree.grantorThe University of Texas at Austinen
thesis.degree.levelDoctoralen
thesis.degree.nameDoctor of Philosophyen


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