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.Science, Faculty ofChemistry, Department ofGraduat