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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
Identification of Two Essential Glutamic Acid Residues in
Glycogen Synthase*
Emili
Cid §,
Roger R.
Gomis ¶,
Roberto A.
Geremia ,
Joan J.
Guinovart , and
Juan C.
Ferrer **
From the 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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Copyright © 2000 by the American Society for Biochemistry and Molecular Biology.
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