A critical inter‐subunit interaction for the transmission of the allosteric signal in the Agrobacterium tumefaciens ADP ‐glucose pyrophosphorylase

Abstract

ADP-glucose pyrophosphorylase is a key regulatory enzyme involved in starchand glycogen synthesis in plants and bacteria, respectively. It has been hypothesized that inter-subunit communications are important for the allosteric effectin this enzyme. However, no specific interactions have been identified as partof the regulatory signal. The enzyme from Agrobacterium tumefaciens is ahomotetramer allosterically regulated by fructose 6-phosphate and pyruvate.Three pairs of distinct subunit-subunit interfaces are present. Here we focuson an interface that features two symmetrical interactions between Arg11 andAsp141 from one subunit with residues Asp141 and Arg11 of the neighbor subunit, respectively. Previously, scanning mutagenesis showed that a mutation atthe Arg11 position disrupted the activation of the enzyme. Considering the distance of these residues from the allosteric and catalytic sites, we hypothesizedthat the interaction between Arg11 and Asp141 is critical for allosteric signaling rather than effector binding. To prove our hypothesis, we mutated thosetwo sites (D141A, D141E, D141N, D141R, R11D, and R11K) and performedkinetic and binding analysis. Mutations that altered the charge affected theregulation the most. To prove that the interaction per se (rather than the presence of specific residues) is critical, we partially rescued the R11D protein byintroducing a second mutation (R11D/D141R). This could not restore the activator effect on kcat, but it did rescue the effect on substrate affinity. Our resultsindicate the critical functional role of Arg11 and Asp141 to relay the allostericsignal in this subunit interface.