Unraveling the activation mechanism for the potato tuber ADP-glucose pyrophosphorylase

FIGUEROA CM; KUHN ML; BALLICORA MA; IGLESIAS AA

Villa Carlos Paz

Congreso; XLIV Reunión Anual de la SAIB; 2008

SAIB

ADPGlcPPase is the regulatory enzyme of glycogen and starch synthesis in bacteria and plants, respectively. The enzyme from plants is a heterotetramer comprised of two small (S, catalytic) and two large (L, regulatory) subunits. Most of the plant ADPGlcPPases are allosterically regulated by 3PGA and Pi. Previous studies on ADPGlcPPase from bacteria pointed out a region critical for activation in the N-term domain. To evaluate if conserved residues in such a region play a role in the potato tuber enzyme, the mutants SQ75A, SW116A, LQ86A and LW128A were constructed, and the different heterotetramers were characterized. Results showed that the mutant enzymes exhibited essentially the same substrate kinetics than the wild type (S/L) enzyme; but the formers were altered in their response to 3PGA. The S/L enzyme was activated 68-fold by 3PGA and inhibited by Pi in a way reverted by the activator. Conversely, for the mutant enzymes S/LQ86A, S/LW128A, SQ75A/L, SW116A/L and SW116A/LW128A activation folds were calculated in 57, 22, 12, 7 and 7, respectively; and Pi inhibition was not modified by 3PGA. This is the first time that the N-term domain of a plant ADPGlcPPase is associated with 3PGA activation. Results agree with a model where the modified residues are located in loops that are responsible for propagating the allosteric activation of the enzyme.