The two activities, glucosidase and transferase, of glycogen debranching enzyme are involved in the degradation of glycogen. The glucosidase site was shown to hydrolyze the substrate α-D-glucosyl fluoride with net inversion of the anomeric configuration. Glycosyl fluorides were shown to be good substrates for the glucosidase and transferase sites, with kinetic parameters k[sub cat] = 1104 min⁻¹ and K[sub m] = 4.2 mM for α-D-glucosyl fluoride with the glucosidase activity, and k[sub cat] = 44

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... -1 and K[sub m] = 11 mM for α-maltotriosyl fluoride in the presence of 1.0% glycogen with the transferase activity. 4-Deoxy-α-maltotriosyl fluoride was found to be an incompetent substrate of the transferase activity as the enzyme would carry out the first step in the double displacement mechanism, glycosylation of the enzyme and release of fluoride ion, but is unable to perform the second step, transfer onto another molecule of itself, because the 4-hydroxy group has been removed. This was shown by the accumulation of a glycosyl-enzyme intermediate as demonstrated by the release of one equivalent of fluoride ion, corresponding to one turnover. Tandem electrospray mass spectrometric (MS/MS) analysis of a proteolytic digest of enzyme reacted with 4-deoxy-α-maltotriosyl fluoride demonstrated that the trisaccharide was covalently attached to a peptide. Subsequent MS/MS experiments on this peptide, along with sequence alignments permitted the identification of the catalytic nucleophile of the transferase activity as aspartic acid 549. Human pancreatic α-amylase is involved in the degradation of starch into simple sugars in the gut. α-Amylase was shown to have an active site composed of five subsites by the kinetic evaluation and determination of the enzymes "action pattern" with the malto-oligosaccharides, maltotetraose through maltoheptaose, using a novel HPLC method. A Dextropak® HPLC column from Waters® was used, which allowed the determination of the stereochemical outcome of the reaction catalyzed by α-amyla [...]