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UBC Theses and Dissertations

Covalent probes for functional and structural characterization of glycoside hydrolases Jain, Namrata


Carbohydrates are ubiquitous in Nature and fundamental to the sustenance of organisms across all domains of life. Carbohydrates serve as sources and reserves of metabolic energy, participate in various cellular communication events, and provide structural support to plant and animal cells. Highly specific enzymes have evolved over several millennia to bind and manipulate carbohydrate substrates. Glycoside hydrolases (GHs) are a class of carbohydrate-active enzymes that cleave glycosidic linkages in complex carbohydrates. Organisms across all domains of life dedicate part of their genome to the production of GHs. New GHs are continually discovered through genome sequencing, while their structural and functional characterization, particularly in complex native environments, poses a persistent challenge to the dynamic field of GH characterization. One of the fundamental ways of ascribing protein function is the exploration of protein active sites, which can be used to deduce important details regarding substrate-enzyme interactions. The work presented in this thesis describes the development of six new probes targeting a variety of GHs by irreversible covalent inhibition. These probes, developed on oligosaccharide scaffolds, feature either an N-bromoacetylglycosylamine electrophilic warhead or 2', 4' dinitrophenyl 2-deoxy-2-fluoro substitutions, facilitating irreversible inhibition of the target GH. The analysis presented in this work reveals key information about enzyme-inhibitor interactions through enzyme kinetic analyses, intact-protein mass spectrometry, and inhibitor-bound protein X-ray crystallography. Enzymes of diverse GH families and substrate preferences including endo-xyloglucanases, mixed-linkage glucanases, and β-(1,3) glucanases are featured to demonstrate the potency of this library of inhibitors. This small-molecule inhibitor toolkit targeting specific GH enzymes has the potential to enhance our knowledge of the structural and functional characteristics of GHs and to provide a platform for activity-based enzyme profiling.

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