Amylo-1,6-glucosidase/1,4-α-glucan: 1,4-α-glucan 4-α-glycosyltransferase: Specificity toward polysaccharide substrates

The substrate specificities of the glucosidase-transferase debranching enzyme systems from yeast and rabbit muscle were examined by the use of polysaccharide substrates of defined outer chain lengths. The results were consistent with the specificities ascribed to the transferase portion of the debranching enzyme system by previous studies using maltosaccharide substrates. The specificities of the two enzyme systems were also examined in the reversion reaction. The results showed that both systems displayed an inverse specificity to that observed in the hydrolytic reaction. This suggested that the reversion reaction reflects the specificity of the glucosidase portion of the debranching system. The major differences between the specificities of the yeast and rabbit muscle systems were found to lie in the specificity of the transferase and in the ability of the yeast system to debranch native glycogen and amylopectin.     

Studies of the residual glycogen branching enzyme activity present in human skin fibroblasts from patients with Type IV glycogen storage disease

Human skin fibroblasts from patients with Type IV glycogen storage disease, in which there is a demonstrable deficiency of glycogen branching enzyme, were shown to be able to synthesize [¹⁴C]glycogen containing [¹⁴C]glucose at branch points when sonicates containing endogenous glycogen synthase $\text{a}$ were incubated with UDP[¹⁴C]glucose. The branch point content of the glycogen synthesized by the Type IV cells was essentially the same as that formed by normal cells, but the total synthetic capacity of the Type IV cells was lower. A new assay for the branching enzyme using glycogen synthase as the indicator enzyme has been developed. Using this assay it has been shown that the residual branching enzyme of affected children and of their heterozygote parents is less easily inhibited by an IgG antibody raised in rabbits against the normal human liver enzyme than is the branching enzyme of normal fibroblasts.     

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.     

Activation of Enzymes during Germination: Amylopectin-1,6-glucosidase in Peas

Development of amylopectin-1, 6-glucosidase (amylopectin 6- glucanohydrolase) activity in germinating peas occurs under conditions in which protein synthesis is inhibited and α- and β-amylase fail to increase in activity. An increase in activity of amylopectin 6-glucanohydrolase can be obtained in cell free homogenates of imbibed pea seeds under autolytic conditions. Activation is shown to occur in a particulate fraction and to be due to the action of a proteolytic enzyme in the homogcnates. A system could be constituted consisting of a particulate fraction and trypsin which lead to a rapid activation of the amylopectin 6-glucanohvdrolase. This is regarded as proof that enzymes in germinating seeds can be formed by liberation or activation of precursor forms. The implications of this are discussed.