Unraveling the Regulation Mechanism for the Potato Tuber ADPglucose Pyrophosphorylase

C.M. FIGUEROA, M.L. KUHN, M.A. BALLICORA, A.A. IGLESIAS

Villa Carlos Paz, Córdoba, Argentina

Congreso; XLIII Reunión Anual de SAIB; 2008

SAIB

UNRAVELING THE REGULATION MECHANISM FORT H E P O TATO T U B E R A D P - G L U C O S EPYROPHOSPHORYLASEFigueroa CM1, Kuhn ML2, Ballicora MA2, Iglesias Aa11Laboratorio de Enzimología Molecular, FBCB, UNL, Argentina &2Department of Chemistry, LUC, USA. E-mail:carfigue@fbcb.unl.edu.arADPGlcPPase is the regulatory enzyme of glycogen and starchsynthesis in bacteria and plants, respectively. The enzyme fromplants is a heterotetramer comprised of two small (S, catalytic) andtwo large (L, regulatory) subunits. Most of the plant ADPGlcPPasesare allosterically regulated by 3PGA and Pi. Previous studies onADPGlcPPase from bacteria pointed out a region critical foractivation in the N-term domain. To evaluate if conserved residuesin such region play a role in the potato tuber enzyme, the mutantsS , S , L and L were constructed, and the different Q75A W116A Q86A W128Aheterotetramers were characterized. Results showed that the mutantenzymes exhibited essentially the same substrate kinetics than thewild type (S/L) enzyme; but the formers were altered in theirresponse to 3PGA. The S/L enzyme was activated 68-fold by 3PGAand inhibited by Pi in a way reverted by the activator. Conversely,for the mutant enzymes S/L , S/L , S /L, S /L and Q86A W128A Q75A W116AS /L activation folds were calculated in 57, 22, 12, 7 and 7, W116A W128Arespectively; and Pi inhibition was not modified by 3PGA. This isthe first time that the N-term domain of a plant ADPGlcPPase isassociated with 3PGA activation. Results agree with a model wherethe modified residues are located in loops that are responsible forpropagating the allosteric activation of the enzyme.