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dc.contributor.authorOsborne, Andrew Stephen
dc.description.abstractChapter I of this dissertation investigates the efficient synthesis of 15N-Ltryptophan, and its subsequent use as an NMR probe for the detection of conformational changes in wild type tryptophan synthase and mutants K87T, D305A, and E109D. 15NHSQC- NMR of enzyme-probe complexes with and without the presence of the a-ligand, a-glycerophosphate, were obtained. The wild type enzyme showed a weak cross peak at 10.25 ppm and 132 ppm in absence of a-glycerophosphate, and a signal six times stronger in the presence of glycerophosphate. Mutants K87T and E109D show a similar cross peak irrespective of a-glycerophosphate, indicating a closed conformation, but mutant D305A only shows a similar peak in the presence of a-glycerophosphate. Chapter II of this dissertation is the report of the efficient and regioselective nitration of Na, N1-bis-trifluoroacetyl-L-tryptophan methyl ester. Aromatic nitration of L-tryptophan has been problematic in the past, and the methodology reported here is useful as the protected nitro-L-tryptophan products are versatile reagents for the synthesis of other aromatic derivatives of L-tryptophan via Sandmeyer chemistry. Both isomers are synthesized in high yield, and purified without chromatography. Chapter III of this dissertation is the kinetic investigation of mutants C295L and C295V of secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus. The mutants were evaluated for their efficiency in catalyzing the oxidation of the small chiral alcohols 2-butanol, 2-pentanol and 2-hexanol at 15º C, 25º C, 35º C, 45º C, and 55º C. Mutant C295L showed a temperature dependence in enantioselectivity for all alcohols, with the (R)-enantiomers being favored at low temperatures. Mutant C295V shows a trend reversal in temperature dependence for 2-butanol and 2-pentanol near 315 K.
dc.subjectTryptophan Synthase
dc.subjectAlcohol Dehydrogenase
dc.subjectThermoanaerobacter ethanolicus
dc.subjectAsymmetric Reduction
dc.titleInvestigations in the chemistry and enzymology of L-tryptophan ; kinetic studies of active site mutants C295L and C295V of secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus
dc.description.advisorRobert S. Phillips
dc.description.committeeRobert S. Phillips
dc.description.committeeGeorge Majetich
dc.description.committeeThomas Johnson

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