Binding of glycogen, oligosaccharides, and glucose to glycogen debranching enzyme.
The binding of glucose and a series of oligosaccharides to glycogen debranching enzyme was determined by the ability of the saccharides to decrease the rate of reaction of sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoate) (DTNB). At pH 7.2, the strength of binding increases with chain length from glucose to maltotriose to maltopentaose but not to maltohexaose, and the free energies for binding of the oligosaccharides suggest subsites of equivalent affinities for the four glucose units following the initial reducing moiety. The rate of reaction of DTNB with enzyme saturated with saccharide is the same for all compounds, suggesting that all the saccharides, including glucose, induce the same conformational state. The site of binding may be that which binds the alpha-1,6-linked side chain of the natural limit dextrin substrate. At pH 8.0, this site exhibits similar characteristics, but an additional site, which may bind the four terminal glucose units of the main chain of the natural substrate, is manifested and exhibits different characteristics, including a very low affinity for glucose itself. The binding of glycogen to the debranching enzyme was monitored by centrifugal separation from the protein and exhibits a much lower dissociation constant than that for the oligomers, suggesting that branched polymers have more than one set of subsites.
Takrama
J
J
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Madsen
N B
NB
D000818:Animals; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism*; D005964:Glucosyltransferases / Q000378:metabolism*; D006003:Glycogen / Q000378:metabolism*; D006004:Glycogen Debranching Enzyme System / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D009844:Oligosaccharides / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000032:analysis
D006004:Glycogen Debranching Enzyme System; D009844:Oligosaccharides; D013438:Sulfhydryl Compounds; D006003:Glycogen; D004228:Dithionitrobenzoic Acid; D005964:Glucosyltransferases; D005947:Glucose