The autogalactosylation of glycogenin

LAFI, IVANA M.; CARRIZO, MARÍA E.; CURTINO, JUAN A.

San Miguel de Tucumán

Congreso; XLV Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB); 2009

Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB)

We have described that glycogenin (GN), at the concentration at which it exists as monomer, has the ability to autoglucosylate with a first order kinetics (Bazán, S. et al., Biochem, Biophys. Res. Commun. 371, 328-332, 2008). In order to understand this intramonomeric mechanism of reaction, a molecular dynamics study will be done, as performed before (Bazán, S. et. al., Biocell 32, 2008), to analyze the distances separating the glucosylable Tyr-194 from Asp-162 (required for activity) and the bound UDPGlc. These distances can not be accurately determined in the reported crystallographic structure of the autoglucosylated GN, because the mobility of the linked maltosaccharide makes "invisible" the Tyr-194 residue. Furthermore, no monoglucosylated GN can be isolated in order to determine such distances to the first incorporated glucose. Here we describe a way to avoid the mobility of the autoglucosylated Tyr-194, and produce a homogeneous monoglycosylated GN species for X-ray diffraction studies, by using UDPGal as donor substrate. The results demonstrate that GN self-incorporates a unique galactose unit into its tyrosine residue, being incapable to continue the O-glycosylation of the linked galactose from UDPGal or UDPGlc.  X-ray diffraction analysis of the obtained galactosylated GN crystals might allow us to determine also the anomeric configuration of the Tyr-O-galactose linkage.