An allosteric mechanism with dual activators regulates the ADP-glucose pyrophosphorylase from Escherichia coli

ASENCIÓN DIEZ, MATÍAS D.; IGLESIAS, ALBERTO A.; BALLÍCORA, MIGUEL A.

Chicago, Illinois

Conferencia; The 33rd Midwest Enzyme Chemistry Conference; 2013

Synthesis of glycogen in bacteria and starch in plants is controlled by ADP-glucose pyrophosphorylase (ADP-Glc ppase), which catalyzes the first committed step in those metabolic pathways. ADP-Glc ppase is an allosterically regulated enzyme with certain promiscuity towards its activators. This is an important evolutionary feature since its preference for regulators varies according to the main metabolic pathway of the organism. Generally, intermediates of those pathways regulate the ADP-Glc ppase. More than nine classes with different specificities have been described. Traditionally, Escherichia coli ADP-Glc ppase has been categorized as mainly activated by Fru-1,6-P2. Here, we found that pyruvate is a key activator that works in a different site, modulating the activity of Fru-1,6-P2. Although pyruvate behaves itself as a modest activator, it acts in synergy with Fru-1,6-P2 to potentiate the fine-tuning regulation of the enzyme, increasing the enzyme affinity for Fru-1,6-P2, and vice versa. The combined action significantly modifies the enzyme kinetic parameters, with an increase in Vmax and a decrease in S0.5 for substrates. Pyruvate also diminishes the inhibitory effect of AMP. The results support the view that pyruvate exerts a fine modulation of glycogen metabolism in E. Coli, by orchestrating a network of allosteric regulators of the enzyme involved in synthesizing the glucosyl donor ADP-glucose. The fact that pyruvate has a synergistic effect on Fru-1,6-P2, implies that they do not compete to bind to the same site. This has a tremendous impact in our understanding of the evolution of allosterism in these enzymes. The presence of more than one allosteric site increases the mutational landscape to explore different regulators and explains the high promiscuity observed in this family. These results do not seem to be restricted to E. Coli. We observed this type of synergistic effects between activators in Agrobacterium tumefaciens ADP-Glc ppase, which belongs to a different class in terms of allosteric specificity (pyruvate and Fru-6P are the main activators). We hypothesize that this pyruvate effect could be dominant or less pronounced in other organisms, depending how significant pyruvate is in the central metabolism, and that evolution has tuned it based on the need of the cell.