Direct detection of glycogenin reaction products during glycogen initiation.
Glycogenin initiates glycogen synthesis in an autocatalytic reaction in which individual glucose residues are covalently linked to Tyrosine 194 in order to form a short priming chain of glucose residues that is a substrate for glycogen synthase which, combined with the branching enzyme, catalyzes the bulk synthesis of glycogen. We sought to develop a new enzymatic assay to better characterize both the chemical and enzymatic characteristics of this unusual reaction. By directly detecting the reaction products using electrospray mass spectrometry this procedure permits both the visualization of the intact individual reaction species produced as a function of time and quantitation of the levels of each of species. The quantitation of the reaction agrees well with previous measurements of both catalytic rate and the change in rate as a function of average glucosylation. The results from this assay provide new insight into the mechanism by which glycogenin catalyzes the initiation reaction.
Hurley
Thomas D
TD
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202-5122, USA. thurley@iupui.edu
Walls
Chad
C
Bennett
John R
JR
Roach
Peter J
PJ
Wang
Mu
M
D002384:Catalysis; D005964:Glucosyltransferases / Q000032:analysis / Q000378:metabolism*; D006003:Glycogen / Q000096:biosynthesis*; D006023:Glycoproteins / Q000032:analysis / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods
D006023:Glycoproteins; C051887:glycogenin; D006003:Glycogen; D005964:Glucosyltransferases