Self-glucosylation of glycogenin, the initiator of glycogen biosynthesis, involves an inter-subunit reaction.
Glycogenin is a dimeric self-glucosylating protein involved in the initiation phase of glycogen biosynthesis. As an enzyme, glycogenin has the unusual property of transferring glucose residues from UDP-glucose to itself, forming an alpha-1,4-glycan of around 10 residues attached to Tyr194. Whether this self-glucosylation reaction is inter- or intramolecular has been debated. We used site-directed mutagenesis of recombinant rabbit muscle glycogenin-1 to address this question. Mutation of highly conserved Lys85 to Gln generated a glycogenin mutant (K85Q) that had only 1-2% of the self-glucosylating activity of wild-type enzyme. Consistent with previous work, mutation of Tyr194 to Phe in a GST-fusion protein yielded a mutant, Y194F, that was catalytically active but incapable of self-glucosylation. The Y194F mutant was able to glucosylate the K85Q mutant. However, there was an initial lag in the self-glucosylation reaction that was abolished by preincubation of the two mutant proteins. The interaction between glycogenin subunits was relatively weak, with a dissociation constant inferred from kinetic experiments of around 2 microM. We propose a model for the glucosylation of K85Q by Y194F in which mixing of the proteins is followed by rate-limiting formation of a species containing both subunit types. The results provide the most direct evidence to date that the self-glucosylation of glycogenin involves an inter-subunit reaction.
Lin
A
A
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122, USA.
Mu
J
J
Yang
J
J
Roach
P J
PJ
D019943:Amino Acid Substitution; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D005964:Glucosyltransferases; D005973:Glutamine / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006003:Glycogen / Q000096:biosynthesis*; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D008239:Lysine / Q000235:genetics; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics
D002148:Calmodulin-Binding Proteins; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C051887:glycogenin; D005973:Glutamine; D014443:Tyrosine; D010649:Phenylalanine; D006003:Glycogen; D005964:Glucosyltransferases; D005982:Glutathione Transferase; D005947:Glucose; D008239:Lysine