Catalytic activities of glycogenin additional to autocatalytic self-glucosylation.
Glycogenin is the autocatalytic, self-glucosylating protein that initiates glycogen synthesis in muscle and other tissues. We have sequenced the cDNA for rabbit muscle glycogenin and expressed and purified the protein in high yield as well as two mutant proteins in which Phe or Thr replaces Tyr-194, the site of glucosylation. While the wild-type protein can self-glucosylate, the mutants cannot, but all three utilize alternative acceptors by intermolecular glucose transfer for which the mutants have altered specificity. Tyr-194 is therefore not essential for the catalytic activity of glycogenin. All three proteins also hydrolyze UDP-glucose to glucose at rates comparable with the rate of self-glucosylation. The hydrolysis is competitive with glucose transfer to p-nitrophenyl alpha-maltoside. Self-glucosylation, glucosylation of other acceptors, and hydrolysis all appear to be catalyzed by the same active center. In the absence of peptidase inhibitors, the homogenous recombinant proteins of M(r) 37,000 break down to equally active species having M(r) 32,000. The kinetics of self-glucosylation catalyzed by the wild-type enzyme suggest that the reaction could be intermolecular rather than, as previously reported, intramolecular. The wild-type recombinant enzyme and native muscle glycogenin, which is phosphorylated, are inhibited quite differently by ATP at physiological concentration.
Alonso
M D
MD
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101, USA.
Lomako
J
J
Lomako
W M
WM
Whelan
W J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D005964:Glucosyltransferases; D006003:Glycogen / Q000096:biosynthesis; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D007700:Kinetics; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*; D015345:Oligonucleotide Probes; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D014443:Tyrosine; D014532:Uridine Diphosphate Glucose / Q000378:metabolism*
D017931:DNA Primers; D006023:Glycoproteins; D009124:Muscle Proteins; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; C051887:glycogenin; D014443:Tyrosine; D006003:Glycogen; D005964:Glucosyltransferases; D014532:Uridine Diphosphate Glucose