Mechanistic Insights into Transition States of Sialidases

Sialic acids, a family of nine carbon sugars, are important components of many biomolecules, and they play important roles in many biological processes. For example, they modulate cellular responses such as differentiation, proliferation and apoptosis. These critical carbohydrates are usually positioned on glycoconjugates as the terminal sugar and they are removed by a family of enzymes called sialidases. In mammals, there are several sialidases that are involved in various biological pathways; however, some human sialidases such as NEU3 have shown to be up-regulated in cancer. Also, certain viruses, bacteria, and trypanosomes have developed sialidases as part of their weaponry. Therefore, it is crucial to design selective and potent inhibitors against these enzymes, with minimal side effects. Development of such selective therapeutics involves a comprehensive understanding of the mechanism by which sialidases catalyze the removal or transfer of sialic acid moieties from glycoconjugates. A key component when studying enzymes mechanisms involves characterization of the transition state(s) (TS) through which the enzyme:substrate complex (ES) is converted to the enzyme:product complex (EP). Hence, the focus of this thesis involves characterization of the transition states (TSs) for sialidase-catalyzed cleavage of alpha-sialosides (sialic acid residues covalently attached to glycoconjugates), by employing three distinct mechanistic tools. These techniques include Brønsted analysis, linear free energy relationship (LFER), and kinetic isotope effects (KIEs). Of note, chapter 4 of this thesis describes the development of a new 2D-NMR technique for measuring multiple kinetic isotope effects simultaneously as a first step in the process of solving transition state structure(s) of sialidase-catalyzed reactions.