Site-directed mutagenesis of lysine382, the activator-binding site, of ADP-glucose pyrophosphorylase from Anabaena PCC 7120.

Previous studies have shown that a highly conserved lysyl residue (Lys 419) near the C-terminus of Anabaena ADP-glucose pyrophosphorylase is involved in the binding of 3-P-glycerate, the allosteric activator [Charng, Y., Iglesias, A. A., & Preiss, J. (1994) J. Biol. Chem. 269, 24107-24113]. Phosphopyridoxylation of the K419R mutant enzyme modified another conserved lysyl residue (Lys382), suggesting that this residue might be also located within the activator-binding site [Charng, Y., Iglesias, AA., & Preiss, J. (1994) J. Biol. Chem. 269, 24107-24113]. Site-directed mutagenesis of Lys382 of the Anabaena enzyme was performed to determine the role of this residue. Replacing Lys382 with either arginine, alanine, or glutamine produced mutant enzymes with apparent affinities for 3-P-glycerate 10-160-fold lower than that of the wild-type enzyme. The glutamic acid mutant enzyme was inhibited by 3-P-glycerate. These mutations had lesser impact on the kinetic constants for the substrates and inhibitor, P(i), and on the thermal stability. These results indicate that both the charge and size of the residue at position 382 influence the binding of 3-P-glycerate. Site-directed mutagenesis was also performed to obtain a K382R-K419R double mutant. The apparent affinity for 3-P-glycerate of this double-mutant enzyme was 104-fold lower than that of the wild-type enzyme, and the specificity for activator of this mutant enzyme was altered. The K382R-K419R enzyme could not be phosphopyridoxylated, suggesting that other lysine residues are not involved in the binding of 3-P-glycerate.