On the simultaneous activation of Agrobacterium tumefaciens ADP-glucose pyrophosphorylase by pyruvate and fructose 6-phosphate.

Bacterial ADP-glucose pyrophosphorylases are allosterically regulated by metabolites that are key intermediates of central pathways in the respective microorganism. Pyruvate (Pyr) and fructose 6-phosphate (Fru6P) activate the enzyme from Agrobacterium tumefaciens by increasing Vmax about 10- and 20-fold, respectively. Here, we studied the combined effect of both metabolites on the enzyme activation. Our results support a model in which there is a synergistic binding of these two activators to two distinct sites and that each activator leads the enzyme to distinct active forms with different properties. In presence of both activators, Pyr had a catalytically dominant effect over Fru6P determining the active conformational state. By mutagenesis we obtained enzyme variants still sensitive to Pyr activation, but in which the allosteric signal by Fru6P was disrupted. This indicated that the activation mechanism for each effector was not the same. The ability for this enzyme to have more than one allosteric activator site, active forms, and allosteric signaling mechanisms is critical to expand the evolvability of its regulation. These synergistic interactions between allosteric activators may represent a feature in other allosteric enzymes.

A novel dual allosteric activation mechanism of Escherichia coli ADP-glucose pyrophosphorylase: the role of pyruvate.

Fructose-1,6-bisphosphate activates ADP-glucose pyrophosphorylase and the synthesis of glycogen in Escherichia coli. Here, we show that although pyruvate is a weak activator by itself, it synergically enhances the fructose-1,6-bisphosphate activation. They increase the enzyme affinity for each other, and the combination increases Vmax, substrate apparent affinity, and decreases AMP inhibition. Our results indicate that there are two distinct interacting allosteric sites for activation. Hence, pyruvate modulates E. coli glycogen metabolism by orchestrating a functional network of allosteric regulators. We postulate that this novel dual activator mechanism increases the evolvability of ADP-glucose pyrophosphorylase and its related metabolic control.

A Chimeric UDP-glucose pyrophosphorylase produced by protein engineering exhibits sensitivity to allosteric regulators.

In bacteria, glycogen or oligosaccharide accumulation involves glucose-1-phosphate partitioning into either ADP-glucose (ADP-Glc) or UDP-Glc. Their respective synthesis is catalyzed by allosterically regulated ADP-Glc pyrophosphorylase (EC 2.7.7.27, ADP-Glc PPase) or unregulated UDP-Glc PPase (EC 2.7.7.9). In this work, we characterized the UDP-Glc PPase from Streptococcus mutans. In addition, we constructed a chimeric protein by cutting the C-terminal domain of the ADP-Glc PPase from Escherichia coli and pasting it to the entire S. mutans UDP-Glc PPase. Both proteins were fully active as UDP-Glc PPases and their kinetic parameters were measured. The chimeric enzyme had a slightly higher affinity for substrates than the native S. mutans UDP-Glc PPase, but the maximal activity was four times lower. Interestingly, the chimeric protein was sensitive to regulation by pyruvate, 3-phosphoglyceric acid and fructose-1,6-bis-phosphate, which are known to be effectors of ADP-Glc PPases from different sources. The three compounds activated the chimeric enzyme up to three-fold, and increased the affinity for substrates. This chimeric protein is the first reported UDP-Glc PPase with allosteric regulatory properties. In addition, this is a pioneer work dealing with a chimeric enzyme constructed as a hybrid of two pyrophosphorylases with different specificity toward nucleoside-diphospho-glucose and our results turn to be relevant for a deeper understanding of the evolution of allosterism in this family of enzymes.