GLYCOGEN PRIMER: GLYCOGENIN GLUCOSYLATION MECHANISM

SOLEDAD BAZÁN; FEDERICO M. ISSOGLIO; MARÍA ELENA CARRIZO; JUAN A. CURTINO

Mar del Plata, Buenos Aires, Argentina

Congreso; XLIII Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB); 2007

Glycogenin (GN) is a self-glucosyltransferase that initiates glycogen de novo biosynthesis in eukaryotic organisms. Since its crystallographic structure was known (Gibbons et al., 2002) the debate about the reaction mechanism of the enzyme was updated.To study the mechanism of glucose incorporation we used two recombinant GN preparations; apo-GN, devoid of sugar and WT-GN containing 4-6 attached glucoses. Firstly, the oligomerization state of GN was analyzed by gel filtration chromatography. GN eluted as dimer when loaded at high protein concentration and as a monomer at low concentration. The glucopolymerization degree of the protein (one xylose or ~ 11 glucoses) didn’t affect its oligomerization state. We measured the specific initial rate of glucose incorporation dependence upon GN concentration, in the range of monomer-dimer coexistence, with the aim of establish the reaction mechanism at different glucopolymerization degrees. In order to differentiate the first sugar incorporation of the following ones we take advantage of the fact that using UDP-xylose as an alternate sugar-donor, GN is capable of incorporate only one sugar molecule (Lomako et al., 1990). The first sugar was incorporated into the tyrosine residue of apo-GN by an intramolecular reaction, with slightly higher catalytic efficiency by monomeric than dimeric GN.  The tyrosine residue of apo-GN is xylosylated at a lower rate than the already partially glucosylated WT-GN. The intermediate growth, until formation of an 4-7 glucose malltosaccharide,  was best achieved by intramolecular transglucosylation between the subunits of dimeric GN. The final growth of the GN-bound malltosaccharide occurred at a high specific reaction rate by intramolecular glucosylation of monomeric GN.