Structure-function relationships of cyanobacterial ADP-glucose pyrophosphorylase. Site-directed mutagenesis and chemical modification of the activator-binding sites of ADP-glucose pyrophosphorylase from Anabaena PCC 7120.

Chemical modification studies of spinach leaf ADP-glucose pyrophosphorylase with pyridoxal-P have shown that a highly conserved lysyl residue near the C terminus might be involved in the binding of 3-P-glycerate, the allosteric activator. Site-directed mutagenesis of the corresponding residue (Lys419) of the Anabaena enzyme was performed to determine the role of this conserved residue. Replacing Lys419 with either arginine, alanine, glutamine, or glutamic acid produced mutant enzymes with apparent affinities for 3-P-glycerate, 25-150-fold lower than that of the wild-type enzyme. The mutant enzymes, however, were still activated to a great extent at higher concentrations of activator suggesting that an additional site or residue was involved in the binding of the activator. These mutations caused lesser or no effect on the kinetic constants for the substrates and inhibitor, P(i), as well as on the catalytic efficiency and thermal stability. The results suggest that both the charge and size of lysine residue 419 are required for the proper binding of 3-P-glycerate. Chemical modification of the Anabaena wild-type enzyme with pyridoxal-P indicated that Lys419 was the only lysyl residue modified. We further performed the same experiment on the K419R mutant enzyme and found another lysyl residue, Lys382, was modified. Reductive phosphopyridoxylation of the wild-type and K419R enzymes caused dramatic alteration in allosteric properties. The activator, 3-P-glycerate, and inhibitor, P(i), protected the enzyme from reductive phosphopyridoxylation. The modified enzymes were more active in the absence of 3-P-glycerate and less sensitive to P(i) inhibition.