Identification of functionally important amino-terminal arginines of Agrobacterium tumefaciens ADP-glucose pyrophosphorylase by alanine scanning mutagenesis

Treatment of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase wi th the arginyl reagent phenylglyoxal resulted in complete desensitization t o fructose 6-phosphate (F6P) activation, and partial desensitization to pyr uvate activation. The enzyme was protected from desensitization by ATP, F6P , pyruvate, and phosphate. Alignment studies revealed that this enzyme cont ains arginine residues in the amino-terminal region that are relatively con served in similarly regulated ADP-glucose pyrophosphorylases. To functional ly evaluate the role(s) of these arginines, alanine scanning mutagenesis wa s performed to generate the following enzymes: R5A, R11A, R22A, R25A, R32A, R33A, R45A, and R60A. All of the enzymes, except R60A, were successfully e xpressed and purified to near homogeneity. Both the R5A and RI IA enzymes d isplayed desensitization to pyruvate, partial activation by F6P, and increa sed sensitivity to phosphate inhibition. Both the R22A and R25A enzymes exh ibited reduced V-max values in the absence of activators, lower apparent af finities for ATP and F6P, and reduced sensitivities to phosphate. The prese nce of F6P restored R22A enzyme activity, while the R25A enzyme exhibited o nly similar to1.5% of the wild-type activity. The R32A enzyme displayed an similar to 11.5-fold reduced affinity for F6P while exhibiting behavior ide ntical to that of the wild type with respect to pyruvate activation. Both t he R33A and R45A enzymes demonstrated a higher activity than the wild-type enzyme in the absence of activators, no response to F6P, partial activation by pyruvate, and desensitization to phosphate inhibition. These altered en zymes were also insensitive to phenylglyoxal. The data demonstrate unique f unctional roles for these arginines and the presence of separate subsites f or the activators.