Regulation of the ADP-Glucose Pyrophosphorylase: Probing Different Allosteric Activation Signals with Sited Directed Mutations

In bacteria and plants, the synthesis of glycogen and starch is controlled by the ADP-glucose pyrophosphorylase (ADP-Glc PPase). The enzyme is activated by fructose-1,6-bisphosphate (FBP) from Escherichia coli and by Pyruvate and F6P from A. tumefaciens, but inhibited by AMP. Previously, we found that some critical amino acids (Trp, Tyr) did not prevent the binding of FBP or F6P, but blocked the activation signal. Considering that two different effectors activate the enzyme, with two different pathways. We investigated whether both allosteric signals follow the path through these critical residues. we recently found that some mutations activate the enzyme by either mimicking the presence of pyruvate or not. Here, the introduction of a second activated mutation also increased the activity of both mutants that blocked the activity when it is single. This indicated that the permanent activation signal created by the active mutant(pre-activated) follows a path that could the other single mutants dependent or independent. We postulate that pyruvate and (FBP or F6P) bind to different sites sending two different allosteric activation signals. Interestingly, the A. tumefaciens. crystal structure that we got has proved the prediction of the other activator binding sit for F6P. In addition, we investigate the activators\u27 binding site from cyanobacteria (Anabaena and Melainabacteria). The data demonstrate unique functional roles for universal arginines for activators and inhibitors. Considering that the two Arg are conserved in very divergent ADP-Glc PPases from bacteria and plants. it seems they anchor the phosphate moiety of any sugar-phosphate activator. We propose the sub-site for binding the phosphate group is conserved, while the residues in charge of recognizing the carbohydrate have diverged