Effects of oligosaccharide binding on glycogen debranching enzyme activity and conformation.
Glycogen debranching enzyme contains two catalytic activities (4-alpha-glucanotransferase and amylo-1,6-glucosidase) on its single polypeptide chain, and they are affected differently by the binding of oligosaccharides. Glucose, maltose, and maltotriose are competitive inhibitors of the amylo-1,6-glucosidase activity measured by the hydrolysis of alpha-glucosyl fluoride, whereas saccharides with four or more glucose units are activators of the same activity, showing apparent "uncompetitive" kinetics. This suggests that they do not bind until the alpha-glucosyl fluoride is bound. In either case the potency of the effect increases with the length of the oligosaccharide chain. On the other hand, all oligosaccharides tested (maltose to maltohexaose, alpha-cyclodextrin, and beta-cyclodextrin) are competitive inhibitors of the transferase activity and also cause a decrease in the intrinsic fluorescence, both functions again increased by chain length, thus indicating that these saccharides do bind to the free enzyme. These interesting results can be reconciled if the extended main chain resulting from the transferase reaction has to be reoriented into a different binding mode in order to position the alpha-1,6-linked side-chain glucose into the correct position for the glucosidase reaction. Therefore, activating oligosaccharides behave kinetically as if they had not been previously bound. It is concluded that the main chain of the natural limit dextrin substrate has a different mode of binding for the two catalytic reactions in order to position properly first the maltotetraosyl side chain in the transferase catalytic site and then the glucosyl side chain in the glucosidase catalytic site.(ABSTRACT TRUNCATED AT 250 WORDS)
Liu
W
W
Department of Surgery, School of Medicine, University of New Mexico, Albuquerque 87131, USA.
Madsen
N B
NB
Fan
B
B
Zucker
K A
KA
Glew
R H
RH
Fry
D E
DE
D000818:Animals; D002942:Circular Dichroism; D005947:Glucose / Q000378:metabolism; D005959:Glucosidases / Q000378:metabolism; D006003:Glycogen / Q000378:metabolism; D006004:Glycogen Debranching Enzyme System / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D009844:Oligosaccharides / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D006004:Glycogen Debranching Enzyme System; D009844:Oligosaccharides; D006003:Glycogen; D005959:Glucosidases; D005947:Glucose