The biogenesis of glycogen involves a specific initiation event mediated by the initiator protein, glycogenin, which undergoes self-glucosylation to generate an oligosaccharide primer from which the glycogen molecule grows. Rabbit muscle glycogenin was expressed at high levels in Escherichia coli and purified close to homogeneity in a procedure that involved binding to a UDP-agarose affinity column. The resulting protein had subunit molecular weight of 38,000 as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Analysis of peptide fragments by mass spectroscopy indicated that the recombinant glycogenin was already glucosylated at Tyr-194 and contained from 1 to 8 glucose residues attached. The enzyme was active as a glucosyl transferase and could incorporate a further ∼5 mol of glucose/mol. The apparent Km for the glucosyl donor UDP-glucose was 4.5 μM, and the pH optimum was pH 8. Of a number of nucleotides and related compounds surveyed, UDP and UTP were the most effective inhibitors. There was also a correlation between inhibition and the presence of a pyrophosphate group. Of several oligosaccharides of glucose, only maltose caused significant inhibition. The glucosylation reaction was first order with respect to glycogenin suggesting that it was intramolecular. The efficacy of the purified glycogenin as a substrate for the elongation reaction catalyzed by glycogen synthase was significantly enhanced if glycogenin was first allowed to undergo self-glucosylation. The length of the priming oligosaccha-ride is thus important for glycogen synthase action. A mutant of glycogenin, in which Tyr-194 was changed to Phe, behaved identically to the wild-type through purification and in particular bound to the UDP-agarose affinity matrix. Despite these indications of the protein's overall structural integrity, it was unable to self-glucosylate. This result indicates that Tyr-194 is necessary for glycogenin function and is consistent with Tyr-194 being the sole site of glucosylation.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - 1993|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology