An Allosteric Effect that enhances Substrate Specificity in the ADP-Glc PPase from Escherichia coli

EBRECHT, A.C.; SOLAMEN, L.; IGLESIAS, A.A.; OLSEN, K.; BALLICORA, M.A.

Congreso; 34th Midwest Enzyme Chemistry Conference; 2014

ADP-Glc PPase is a key enzyme responsible for the production of ADP-Glc, the sugar nucleotide used in the synthesis of glycogen and starch in bacteria and plants, respectively1,2. The enzyme activity is allosterically modulated by various metabolites that indicate the energy levels of the cell. Thus, the ADP-Glc PPase is the regulatory enzyme of the pathway1,2. In particular the E. coli ADP-Glc PPase is mainly activated by fructose-1,6-P2 (FBP). Moreover, pyruvate (Pyr) behaves itself as a modest activator but it acts in synergy with FBP to finely modulate enzyme activity3. We observed that this enzyme exhibits a degree of promiscuity toward the substrates and the essential cofactor. Interestingly such a promiscuous behavior was found affected by the allosteric activators in a way that they markedly increased the affinity of the enzyme toward the use of ATP, but not of other NTPs. The "selectivity" effect of FBP was also studied respect to the sugar-1P substrate as well on the essential metal ion required for catalysis. The allosteric activator increased catalytic efficiency of the enzyme for Glc-1P>>GlcN-1P>Gal-1P. Under all of the conditions (plus or minus allosteric activator) the greatest catalytic efficiency was achieved with Glc-1P as substrate. Co2+ and Mn2+ behaved as cofactors in reactions with both ATP and UTP. Although only in the reactions performed with ATP the FBP increased the catalytic efficiency. These results support a view in which the allosteric regulator would play a critical role in the specific selection of ATP as a substrate, enhancing the production of the sugar nucleotide that leading to synthesis of glycogen in vivo. This view of the effectors as a mechanism to increase the specificity of the enzyme opens new perspectives for the understanding of the allosteric regulation mechanisms of ADP-Glc PPases (and possibly other enzymes) in the context of the metabolic scenario.