Identification of Asp 549 as the catalytic nucleophile of glycogen-debranching enzyme via trapping of the glycosyl-enzyme intermediate.
Glycogen-debranching enzyme catalyzes the removal of branching from glycogen via a two-step process involving first the transfer of a maltotriosyl unit from the branch to the main chain and second the hydrolysis of the residual alpha-(1,6)-linked glucose moiety. Since the transfer occurs with retention of anomeric configuration, a mechanism involving a maltotriosyl-enzyme species is presumed. 4-Deoxy-alpha-maltotriosyl fluoride functions as an incompetent substrate for this transferase activity since a glycosyl-enzyme species in formed, as witnessed by a "burst" of fluoride release, but turned over only very slowly unless a suitable acceptor such as maltotriose is added, at which point 4-deoxymaltohexaose is released. Peptic proteolysis of this trapped enzyme generated a mixture of peptides which was separated by reverse phase high-performance liquid chromatography, and the glycosylated peptide was located by use of tandem mass spectrometry in the neutral loss mode. Subsequent tandem mass spectrometric experiments on this peptide identified it as one surrounding Asp 549. This amino acid is completely conserved in all alpha-glucanotransferases and alpha-glucosidases belonging to this sequence -related family and is hereby identified as the catalytic nucleophile.
Braun
C
C
Department of Chemistry, University of British Columbia, Vancouver, Canada.
Lindhorst
T
T
Madsen
N B
NB
Withers
S G
SG
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry*; D002240:Carbohydrate Sequence; D002384:Catalysis; D016384:Consensus Sequence; D006003:Glycogen / Q000378:metabolism; D006004:Glycogen Debranching Enzyme System / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D011817:Rabbits; D017386:Sequence Homology, Amino Acid
D006004:Glycogen Debranching Enzyme System; D001224:Aspartic Acid; D006003:Glycogen