Biochemical studies on plant ADP-glucose pyrophosphorylase regulatory properties

ADP-glucose pyrophosphorylase (AGPase) catalyzes the first 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 wild type SS, formed 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. Several putative SS mutant suppressors were identified by their ability to restore glycogen accumulation when co-expressed with LSP52L mutant in Escherichia coli glgC-strain. Kinetic analysis of these second-site mutant SS enzymes indicated that they comprise two distinct classes based on the SS interaction with LSP52L or wild type LS. One class contained bona fide SS suppressors (SS L46F and SSP112L), which reversed the down-regulatory properties of LSP52L but not to wild type LS. The other SS class contained allosteric mutants, SSP308L and SSR350K, which generated up-regulated enzymes with wild type LS as well as LSP52L. These results indicate that both LS and SS have a regulatory function in controlling allosteric properties through enhancing subunit interactions. In addition to allosteric regulation, plant AGPases are redox regulated by reduction of an intermolecular disulfide bond (S-S) between two Cys12 residues of catalytic small subunits (SSs). In this study, we replaced the Cys 12 residues with Ala (SSC12A) to remove the disulfide bond in both the potato tuber and Arabidopsis leaf AGPase SSs. SSC12As were co-expressed with the corresponding LSs and resulting enzymes were purified. Kinetic analysis of SSC12A containing enzymes indicated that they have high affinity to the activator 3-phosphoglycerate and substrate ATP in both reducing and oxidizing conditions. Thermal stability of these enzymes was also investigated using both kinetics and biochemical approaches. SS C12A containing enzymes were less stable than the wild type enzymes at high temperatures, based on the results from circular dichroism spectroscopy and enzyme activity. Our results suggested the S-S bond in the AGPase is important for not only enhancing enzyme activation through higher affinity to activator and substrate, but also enzyme stability to maintain ordered structure at high temperatures.