Identification of Asp 549 as the catalytic nucleophile of glycogen-debranching enzyme via trapping of the glycosyl-enzyme intermediate

Biochemistry
C BraunS G Withers

Abstract

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 identi...Continue Reading

References

Mar 1, 1984·Journal of Biochemistry·Y MatsuuraM Kakudo
Jul 10, 1995·Carbohydrate Research·C Braun, S G Withers
Dec 1, 1994·Current Opinion in Structural Biology·J D McCarter, S G Withers
Mar 1, 1995·Protein Science : a Publication of the Protein Society·S G Withers, R Aebersold
Oct 1, 1993·Archives of Biochemistry and Biophysics·W LiuR C Bleackley

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Citations

Sep 1, 2000·Carbohydrate Research·S J Williams, S G Withers
Jan 1, 1997·Progress in Biophysics and Molecular Biology·S Janecek
Mar 21, 2001·Biochimica Et Biophysica Acta·E A MacGregorB Svensson
Oct 20, 2005·Journal of Bioscience and Bioengineering·T Kuriki, T Imanaka
Mar 26, 1999·Protein Science : a Publication of the Protein Society·E H RydbergS G Withers
Jun 29, 2000·Annual Review of Biochemistry·H D Ly, S G Withers
May 29, 2001·The Journal of Biological Chemistry·A NakayamaS Tabata
May 20, 2000·Biotechnology & Genetic Engineering Reviews·T Takaha, S M Smith

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