Role of the inter‐subunit surface interaction in the regulation of the ADP‐glucose pyrophosphorylase from Agrobacterium tumefaciens

Abstract

ADP‐glucose pyrophosphorylase (ADP‐Glc PPase) is a key regulatory enzyme involves in the starch and glycogen synthesis for plants and bacteria respectively, which catalyzes the synthesis of ADP‐glucose from glucose 1‐phosphate (Glc1P) and ATP. ADP‐Glc PPase from Agrobacterium tumefaciens is allosterically regulated by fructose 6‐phosphate (Fru6P) and pyruvate (Pyr). Previously, it was observed that removal of a disulfide bridge between the α‐subunits of the potato tuber enzyme resulted in a more active enzyme [1], indicating that inter‐subunit interactions are important in the allosteric mechanism [2]. By studying the structure of the ADP‐Glc PPase enzyme of Agrobacterium tumefaciens, it comes to light that D141 is interacting with the R11 of the opposing subunit [3], which was suggested to be involved in activation by pyruvate [4]. To prove our hypothesis that D141 and R11 residues are involved in regulation, we made three single mutants: D141A, D141R, R11D, and the double mutant R11D‐D141R. D141A exhibited reduced Vmax values in the absence of activators (~4‐fold), reduced apparent affinity for Fru6P (~2.5‐fold), and lower apparent affinity for ATP (~3‐fold). In the presence of Fru6P, D141A exhibited only ~4.3% of the wild‐type activity, and affinity for ATP was not affected as well indicating a virtual insensitivity to Fru6P. When D141R was compared to wild‐type enzyme, the activity lowered ~3600‐fold. For D141R the apparent affinity for ATP was decreased in the presence of Fru6P. R11D was similarly active as the wild‐type, while it is completely insensitive to both activators Fru6P and Pyr. The double mutant R11D‐D141R was showing decreased activity (~95‐fold) in compared to wild type, and insensitive to both activators. Our results suggest that the functional role of the D141 and R11 residues is to interacting with the opposing subunit and enabling the regulation of the ADP‐Glc PPase.Support or Funding InformationNational Science Foundation