Characterization of rabbit skeletal muscle glycogenin: Tyrosine 194 is essential for function

Youjia Cao, Alan M. Mahrenholz, Anna De Paoli-Roach, Peter Roach

Research output: Contribution to journalArticle

57 Citations (Scopus)

Abstract

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 languageEnglish
Pages (from-to)14687-14693
Number of pages7
JournalJournal of Biological Chemistry
Volume268
Issue number20
StatePublished - Jul 15 1993

Fingerprint

Tyrosine
Muscle
Skeletal Muscle
Rabbits
Uridine Diphosphate
Glycogen Synthase
Oligosaccharides
Glycogen
Glucose
Sepharose
Uridine Diphosphate Glucose
Uridine Triphosphate
Peptide Fragments
Proteins
Maltose
glycogenin
Protein Subunits
Structural integrity
Transferases
Electrophoresis

ASJC Scopus subject areas

  • Biochemistry

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Characterization of rabbit skeletal muscle glycogenin : Tyrosine 194 is essential for function. / Cao, Youjia; Mahrenholz, Alan M.; De Paoli-Roach, Anna; Roach, Peter.

In: Journal of Biological Chemistry, Vol. 268, No. 20, 15.07.1993, p. 14687-14693.

Research output: Contribution to journalArticle

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