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Originally published In Press as doi:10.1074/jbc.M005358200 on August 2, 2000

J. Biol. Chem., Vol. 275, Issue 43, 33614-33621, October 27, 2000
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Identification of Two Essential Glutamic Acid Residues in Glycogen Synthase*

Emili CidDagger §, Roger R. GomisDagger , Roberto A. Geremia||, Joan J. GuinovartDagger , and Juan C. FerrerDagger **

From the Dagger  Departament de Bioquímica i Biologia Molecular, Universitat de Barcelona, Barcelona E-08028, Spain and the || Centre des Recherches sur les Macromolécules Végétales, CNRS, affiliated with the Joseph Fourier University, Grenoble F-38041, France

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-X7-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-X7-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.

* This work was supported in part by Grant PM98-0185 from Dirección General de Enseñanza Superior (Ministerio de Educación y Cultura, Spain), by Grant ACI 99-16 from the Generalitat de Catalunya, and by the Juvenile Diabetes Foundation International.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Recipient of a doctoral fellowship from the Generalitat de Catalunya (Comissió Interdepartamental de Recerca i Innovació Tecnològica).

Recipient of a doctoral fellowship (Formación Personal Investigador) from the Spanish Government (Ministerio de Educación y Cultura).

** To whom correspondence should be addressed: Dept. de Bioquímica i Biologia Molecular, Universitat de Barcelona, Martí i Franquès, 1, Barcelona E-08028, Spain. Tel.: 34-93-402-1209; Fax: 34-93-402-1219; E-mail: ferrer@sun.bq.ub.es.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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