Probing Allosteric Binding Sites of the Maize Endosperm ADP-Glucose Pyrophosphorylase

Abstract

Abstract Maize (Zea mays) endosperm ADP-glucose pyrophosphorylase (AGPase) is a highly regulated enzyme that catalyzes the rate-limiting step in starch biosynthesis. Although the structure of the heterotetrameric maize endosperm AGPase remains unsolved, structures of a nonnative, low-activity form of the potato tuber (Solanum tuberosum) AGPase (small subunit homotetramer) reported previously by others revealed that several sulfate ions bind to each enzyme. These sites are also believed to interact with allosteric regulators such as inorganic phosphate and 3-phosphoglycerate (3-PGA). Several arginine (Arg) side chains contact the bound sulfate ions in the potato structure and likely play important roles in allosteric effector binding. Alanine-scanning mutagenesis was applied to the corresponding Arg residues in both the small and large subunits of maize endosperm AGPase to determine their roles in allosteric regulation and thermal stability. Steady-state kinetic and regulatory parameters were measured for each mutant. All of the Arg mutants examined—in both the small and large subunits—bound 3-PGA more weakly than the wild type (A  50 increased by 3.5- to 20-fold). By contrast, the binding of two other maize AGPase allosteric activators (fructose-6-phosphate and glucose-6-phosphate) did not always mimic the changes observed for 3-PGA. In fact, compared to 3-PGA, fructose-6-phosphate is a more efficient activator in two of the Arg mutants. Phosphate binding was also affected by Arg substitutions. The combined data support a model for the binding interactions associated with 3-PGA in which allosteric activators and inorganic phosphate compete directly.