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UBC Theses and Dissertations

Mechanisms of alpha-glycosidases Numao, Shin


α-Retaining glycosidases are a class of hydrolytic enzymes that cleave α-glycosidic linkages with net retention of configuration at the anomeric center. The finer details of the mechanisms of two such enzymes, Golgi α-mannosidase II and human pancreatic α-amylase, were investigated in order to gain better understanding of this class of enzymes. α-Retaining mannosidases, like all other retaining glycosidases, employ a double displacement mechanism in which a transient glycosyl-enzyme intermediate is formed. In order to isolate or "trap" this key intermediate, two active site probes were synthesized for family 38 α-mannosidases. Both 5-fluoro-β-L-gulosyl fluoride and 2-deoxy-2-fluoro- α- D-mannosyl-fluoride were shown to be poor substrates for Golgi α-mannosidase II at room temperature. In both cases, by lowering the temperature, the lifetime of the intermediate increased so that the species was experimentally observable. Structural studies using X-ray crystallography showed that in both cases, the intermediate sugars adopted a ¹S₅ skew boat conformation, suggesting that the intermediate formed during the hydrolysis of the natural substrate also adopts this conformation. This suggests that intermediate distortion is an important part of the catalytic mechanism in α -glycosidases. α-Amylases are a family of α-retaining glycosidases involved in glucose polymer digestion. Several members of this family are activated in the presence of chloride ion. To investigate the mechanism of this phenomenon, a series of mutations were made to the residues constituting the chloride ion-binding site in human pancreatic a-amylase (HPA). Kinetic analysis of the resultant mutants showed that one of these residues, Arg337, in the absence of chloride, effectively inhibits the enzyme by modulating the pK[sub a] of the acid/base catalyst. The chloride ion activates HPA by relieving this inhibition through charge shielding. Upon mutation of this residue, the mutant HPA became a chloride-independent enzyme with full catalytic activity. Using a novel substrate, 2,4-dinitrophenyl α-maltotrioside, a new rapid screen was developed to identify potential HPA inhibitors. Using this screen, Dgluconohydroximino- l,5-lactam (GHIL) was determined to be a potential HPA inhibitor. Kinetic analysis of this compound showed that GHIL is a poor inhibitor of HPA with a K[sub i] value of 11 mM. However, in the presence of substrate, HPA will take this compound and modify it to create a more potent inhibitor in situ. This modification is most likely the result of a transglycosylation of the substrate to the inhibitor.

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