Sialic acids are a family of 9-carbon keto sugars found throughout nature. The most prevalent example is N-acetylneuraminic acid (Neu5Ac). This carbohydrate is commonly found capping the terminal ends of a variety of glycoconjugates and polysaccharides. The family of enzymes that catalyze the hydrolytic cleavage of Neu5Ac containing complexes are known as sialidases. Numerous studies have implicated the involvement of these enzymes in human diseases such as cancer, cholera and influenza. As such, sialidase inhibitors can function as tools to study these conditions or to serve as potential therapeutics. Rational drug design has emerged as a powerful tool used to develop such compounds. This process involves acquiring a comprehensive mechanistic understanding of the target enzyme. Particularly interesting are features manifested at the enzymatic transition state (TS) which include the extent of bond-formation/cleavage, charge development and geometry. Since sialidases operate via a double displacement mechanism, both glycosylation and deglycosylation TSs must be considered to fully understand this family of enzymes. Thus, a major component and objective of this thesis work was to use established techniques such as enzyme kinetics, mutagenesis and the measurement and analysis of multiple kinetic isotope effects (KIEs) to unravel the mechanism for various sialidases. With regard to KIEs, a novel NMR-based technique was developed and employed to measure competitive enzymatic KIEs on the sialidase-catalyzed hydrolysis reactions.
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Thesis advisor: Bennet, Andrew J.
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