CONICET | Buscador de Institutos y Recursos Humanos

ADP-glucose pyrophosphorylase (ADPGlcPPase) catalyzes the regulatory step for glycogen and starch synthesis in bacteria and plants, respectively. The enzyme is allosterically regulated by metabolites that characteristically are key intermediaries of the central carbon pathway in the respective organism. Studies of structure-to-function relationships identified specific loops facing close in the 3D structure of the homotetrameric ADPGlcPPase from bacteria, and which interaction was found critical for propagating the activation signal exerted by Fru1,6bisP. To explore if the latter is a common mechanism, we sought for its occurrence in the plant enzyme, which is hetero-oligomeric and regulated by 3PGA and Pi. Studies were performed with the potato tuber ADPGlcPPase, comprised of small (S, catalytic) and large (L, regulatory) subunits. Conserved residues in specific loops were mutated to construct mutants: Q75A and W116A in the S-subunit, and Q86A and W128A in the L-subunit. The different S2/L2 heterotetramers were kinetically characterized. The mutant enzymes exhibited essentially the same substrate kinetics than the wild type enzyme; but the formers were altered (to different degrees) in their response to allosteric regulators. Thus, the wild type enzyme was activated 68-fold by 3PGA and inhibited by Pi in a way reverted by the activator; whereas mutants exhibited lower activation (between 57- and 7-fold) and lower interaction between the activator and the inhibitor. Molecular modelling analysis reinforced the mutagenesis data, agreeing with a model where dynamics at loops in the N-terminal region are responsible for propagating the allosteric activation of the plant enzyme. The model supports a main regulatory function for the L-subunit in the potato tuber ADPGlcPPase.