Enzymes that catalyze the removal of carbohydrate linkages from biological molecules are called glycosyl hydrolases (GH). These enzymes have been catagorized into more than 130 different families. Family-4 glycosyl hydrolases catalyze hydrolysis via an unusual oxidation-reduction mechanism that requires NAD+, Mn2+ and reducing conditions for catalytic activity, a situation that contrasts the classical nucleophilic substitution mechanism of most glycosyl hydrolases. Most of this thesis is directed towards detailed mechanistic evaluations of the GH4 alpha-galactosidase from Citrobacter freundii and the GH109 alpha-N-acetylgalactosaminidase, which has a bound active site NAD+, from Elizabethkingia meningosepticum. Experimentally, this involved cloning the gene, expressing and purifying the recombinant protein, synthesizing substrates, measuring pH-rate profiles, Brønsted parameters and deuterium kinetic isotope effects (KIEs). The MelA alpha-galactosidase (GH4, C. freundii) possesses a substrate specificity that is limited to non-phosphorylated alpha-D-galactosides. The measured primary deuterium KIEs show that the first irreversible step involves a concerted oxidation of the C-3 hydroxyl group and proton abstraction at C-2. Subsequent elimination of the leaving group is kinetically silent. The GH109 alpha-N-acetylgalactosaminidase (E. meningosepticum) displayed a significant degree of substrate promiscuity as it catalyzed the hydrolysis of aryl 2-acetamido-2-deoxy-alpha-D-galactosides and aryl alpha-D-galactosides efficiently. Notably, Brønsted analysis on both aryl 2-acetamido-2-deoxy-D-galactosides and aryl alpha-D-galactosides suggests that leaving group departure is not rate limiting. The measured deuterium KIEs kinetic isotope effects using the "non-natural" phenyl alpha-D-galactoside substrate revealed that both C-3 oxidation and C-2 proton abstraction are kinetically significant. A bicyclo[4.1.0]heptyl mimic of an aryl alpha-D-galactopyranoside, (1R,2S,3S,4R,5S,6S)-5-(3,5-difluorophenoxy)-1-(hydroxymethyl)bicyclo[4.1.0]heptan-2,3,4-triol, was made and shown to be an irreversible inhibitor of the GH36 alpha-galactosidase from Thermotogamaritima. In contrast, the diastereomeric L-altro isomer failed to inactivate the enzyme, while the analogous cyclohexene was shown to be a tight-binding competitive inhibitor. The measured pH-rate profiles for inhibition and reactivation as well as the corresponding catalytic and inhibitory proficiencies suggested that inhibition results from the formation of an oxacarbenium ion in the enzymatic site that is trapped rapidly by an active site enzymatic residue.
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Thesis advisor: Bennet, Andrew
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