NDLI: Identification of Two Essential Glutamic Acid Residues in Glycogen Synthase

Content Provider	Journal of Biological Chemistry (JBC)
Author	Cid, Emili Gomis, Roger R. Geremia, Roberto A. Guinovart, Joan J. Ferrer, Juan C.
Abstract	The detailed catalytic mechanism by which glycosyltransferases catalyze the transfer of a glycosyl residue from a donor sugar to an acceptor is not known. Through the multiple alignment of all known eukaryotic glycogen synthases we have found an invariant 17-amino acid stretch enclosed within the most conserved region of the members of this family. This peptide includes an E-X 7-E motif, which is highly conserved in four families of retaining glycosyltransferases. Site-directed mutagenesis was performed in human muscle glycogen synthase to analyze the roles of the two conserved Glu residues (Glu-510 and Glu-518) of the motif. Proteins were transiently expressed in COS-1 cells as fusions to green fluorescence protein. The E510A and E518A mutant proteins retained the ability to translocate from the nucleus to the cytosol in response to glucose and to bind to intracellular glycogen. Although the E518A variant had approximately 6% of the catalytic activity shown by the green fluorescence protein-human muscle glycogen synthase fusion protein, the E510A mutation inactivated the enzyme. These results led us to conclude that the E-X 7-E motif is part of the active site of eukaryotic glycogen synthases and that both conserved Glu residues are involved in catalysis. We propose that Glu-510 may function as the nucleophile and Glu-518 as the general acid/base catalyst.
Related Links	http://www.jbc.org/content/275/43/33614.abstract
Ending Page	33621
Starting Page	33614
Page Count	8
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	43
Volume Number	275
DOI	10.1074/jbc.M005358200
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2000-10-27
Access Restriction	Open
Subject Keyword	Glycogen synthase (GS) Human muscle glycogen synthase (HMGS) Green fluorescence protein (GFP) Hydrophobic cluster analysis (HCA) Dulbecco's modified Eagle's medium (DMEM) Fetal bovine serum (FBS) Phosphate-buffered saline (PBS) Bovine serum albumin (BSA) Glucose 6-phosphate (Glu 6-P) Tetramethylrhodamine isothiocyanate (TRITC) Nucleotide recognition domain 1α (NRD1α) Open reading frame (ORF) ENZYME CATALYSIS AND REGULATION
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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