Identification of two essential glutamic acid residues in glycogen synthase

The detailed catalytic mechanism by which glycosyltransferases catalyze the transfer of a glycosyl residue from a donor sugar to an acceptor is not kn own, Through the multiple alignment of all known eukaryotic glycogen syntha ses we have found an invariant 17-amino acid stretch enclosed within the mo st 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 gly cosyltransferases. Site-directed mutagenesis was performed in human muscle glycogen synthase to analyze the roles of the two conserved Glu residues (G lu-510 and Glu-518) of the motif. Proteins were transiently expressed in CO S-l cells as fusions to green fluorescence protein. The E510A and E518A mut ant proteins retained the ability to translocate from the nucleus to the cy tosol in response to glucose and to bind to intracellular glycogen. Althoug h the E518A variant had approximately 6% of the catalytic activity shown by the green fluorescence protein-human muscle glycogen synthase fusion prote in, the E510A mutation inactivated the enzyme. These results led us to conc lude that the E-X-7-E motif is part of the active site of eukaryotic glycog en 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 th e general acid/base catalyst.