Active site mapping of amylo-alpha-1,6-glucosidase in porcine liver glycogen debranching enzyme using fluorogenic 6-O-alpha-glucosyl-maltooligosaccharides.
Glycogen debranching enzyme (GDE) has two enzymatic activities, 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase. Products with 6-O-alpha-glucosyl structures formed from phosphorylase limit dextrin by the 4-alpha-glucanotransferase activity are hydrolyzed to glucose by the amylo-alpha-1,6-glucosidase activity. Here, we probed the active site of amylo-alpha-1,6-glucosidase in porcine liver GDE using various 6-O-alpha-glucosyl-pyridylamino (PA)-maltooligosaccharides, with structures (Glcalpha1-4)(m)(Glcalpha1-6)Glcalpha1-4(Glcalpha1-4)(n)GlcPA (GlcPA, 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol residue). Fluorogenic dextrins were prepared from 6-O-alpha-glucosyl-alpha-, beta-, or gamma-cyclodextrin through partial acid hydrolysis, followed by fluorescent tagging of the reducing-end residues of the hydrolysates and separation by gel filtration and reversed-phase HPLC. Porcine liver GDE hydrolyzed dextrins with the structure Glcalpha1-4(Glcalpha1-6)Glcalpha1-4Glc to glucose and the corresponding PA-maltooligosaccharides, whereas other dextrins were not hydrolyzed. Thus, substrates must have two glucosyl residues sandwiching the isomaltosyl moiety to be hydrolyzed. The rate of hydrolysis increased as m increased and reached maximum at m = 4. The rates were the highest when n = 1 but did not vary much with changes in n. Of the dextrins examined, Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-6)Glcalpha1-4Glcalpha1-4GlcPA (6(3)-O-alpha-glucosyl-PA-maltoheptaose) was hydrolyzed most rapidly, suggesting that it fits the best in the amylo-alpha-1,6-glucosidase active site. It is likely that the active site accommodates 6(2)-O-alpha-glucosyl-maltohexaose and that the interactions of seven glucosyl residues with the active site allow the most rapid hydrolysis of the alpha-1,6-glucosidic linkage of the isomaltosyl moiety.
Yamamoto
Eriko
E
Department of Chemistry, Graduate School of Science, Osaka Prefecture University, 2-1, Daisen-cho, Sakai, Osaka 590-0035, Japan.
Makino
Yasushi
Y
Omichi
Kaoru
K
D000818:Animals; D001665:Binding Sites / Q000502:physiology*; D002240:Carbohydrate Sequence; D003505:Cyclodextrins; D005456:Fluorescent Dyes; D006004:Glycogen Debranching Enzyme System / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D009844:Oligosaccharides / Q000378:metabolism*; D025942:Oligosaccharides, Branched-Chain / Q000378:metabolism; D011725:Pyridines; D034421:Sus scrofa
D003505:Cyclodextrins; D005456:Fluorescent Dyes; D006004:Glycogen Debranching Enzyme System; D009844:Oligosaccharides; D025942:Oligosaccharides, Branched-Chain; D011725:Pyridines; C021705:maltooligosaccharides; C030563:amylo-1,6-glucosidase