Reassessment of the catalytic mechanism of glycogen debranching enzyme.
The amylo-1,6-glucosidase catalytic activity of glycogen debranching enzyme allows it to hydrolyze alpha-D-glucosyl fluoride, in the absence or presence of glycogen or oligosaccharides, releasing equal amounts of fluoride and glucose at rates comparable to those seen with the natural substrates. 2-Deoxy-2-fluoro-alpha-D-glucosyl fluoride is found to be a poor substrate, rather than the covalent inhibitor that would be expected for a glucosidase which catalyzes hydrolysis of the glycosidic linkage with retention of anomeric configuration. In fact, analysis of the glucosidase reaction by NMR reveals that the debranching enzyme hydrolyzes the glycosidic linkage with inversion of configuration, releasing beta-D-glucose from both alpha-glucosyl fluoride and its natural substrate, the phosphorylase limit dextrin. In contrast, its transferase activity necessarily proceeds with retention of configuration. As has been seen with other "inverting" glycosidases, the debranching enzyme releases beta-D-glucose from beta-D-glucosyl fluoride in the presence of oligosaccharides such as maltohexaose and cyclomaltoheptaose but, unlike the others, not in their absence. An intermediate glucosyl-alpha-(1,6)-cyclomaltoheptaose has been detected by NMR analysis. In the presence of a water-soluble carbodiimide, a single mole of glycine ethyl ester is incorporated into each mole of the debranching enzyme, resulting in its inactivation when measured by the combined assay for both transferase and glucosidase activities. Measurement of the latter two activities independently indicates that it is the transferase activity which is inactivated, while the glucosidase activity, measured with alpha-D-glucosyl fluoride as substrate, is unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)
Liu
W
W
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Madsen
N B
NB
Braun
C
C
Withers
S G
SG
D000818:Animals; D001665:Binding Sites; D002234:Carbodiimides / Q000494:pharmacology; D002384:Catalysis; D005459:Fluorides / Q000378:metabolism; D005960:Glucosides / Q000378:metabolism; D006004:Glycogen Debranching Enzyme System / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000648:ultrastructure; D009132:Muscles / Q000201:enzymology; D011817:Rabbits
D002234:Carbodiimides; D005960:Glucosides; D006004:Glycogen Debranching Enzyme System; D005459:Fluorides