Two distinct types of allosteric SS mutants suggest subunit interaction determines allosteric properties of Potato tuber ADP Glucose Pyrophosphorylase

ADP‐glucose pyrophosphorylase (AGPase) catalyzes the committed step of starch synthesis in plants. Plant AGPases are composed of two distinct subunits, a catalytic small subunit (SS) and a non‐catalytic large subunit (LS). Previously, we have identified and characterized an allosteric LS‐P52L mutant, which when co‐expressed with wildtype SS resulted in the formation of an enzyme with down‐regulating allosteric properties. To further investigate the structure‐function relationships between the two subunits with regard to allosteric regulation, random chemical mutagenesis was performed to generate SS suppressors of LS‐P52L. Seven putative SS mutant suppressors were identified by their ability to restore glycogen accumulation when co‐expressed with LS‐P52L in Escherichia coli glgC ‐ strain. Kinetic analysis of four of these second‐site mutant SS enzymes containing LS‐P52L or wildtype LS indicated that they comprise two distinct classes. One class contained bona fide SS suppressors (L48F and P114L), which conferred up‐regulatory properties to LS‐P52L but not to wildtype LS. The other SS class contained allosteric mutants, P310L and R352K, which generated up‐regulated enzymes with wildtype LS as well as LS‐P52L. These results indicate that both LS and SS have a regulatory function in controlling allosteric properties through enhancing subunit interactions either cooperatively or independently.