The allosteric nature of Human Glycogen Branching Enzyme

ISSOGLIO, FEDERICO M.; CARRIZO, MARÍA E.; CURTINO, JUAN A.

Rosario, Santa Fe, Argentina

Congreso; L Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB); 2014

Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB)

Glucose is the principal source of energy for most cells. In mammals and bacteria, glucose is stored as glycogen, the branched polymer formed by linear α-1,4-oligoglucan chains linked by α-1,6-glucosidic bonds. The de novo biosynthesis of glycogen starts with glycogenin autoglucosylation, that produces a protein-bound oligoglucan that serves as primer for other two enzymes, glycogen synthase elongating the chains, and the glycogen branching enzyme (GBE) catalyzing the cleavage of a linear segment and transferring it to the 6-position of a non-terminal glucosyl unit. GBE belongs to the Glycosil Hydrolase family 13, and has been isolated and characterized in some bacteria, rabbit and rat. In this study we report the production of human GBE (HBE) in insect cells using the baculovirus expression system, and the activity analysis of the recombinant enzyme. The branches introduced by HBE were analyzed after degradation with isoamylase. The new reducing oligosaccharides generated are subjected to fluorophore-assisted carbohydrate electrophoresis (FACE), where saccharides are derivatized at their reducing ends with a fluorophore and then separated by polyacrilamide gel electrophoresis (PAGE). Using this method we could measure HBE activity by quantifying the specific reaction products, and perform the kinetic studies that reveal the allosteric nature of this enzyme.