Structure-Function Relationships of Cyanobacterial ADP-glucose Pyrophosphorylase. Site-Directed Mutagenesis and Chemical Modification of the Activator-Binding Sites of ADPglucose Pyrophosphorylase from Anabaena PCC 7120

Y-Y. CHARNG, A.A. IGLESIAS Y J. PREISS

JOURNAL OF BIOLOGICAL CHEMISTRY

Año: 1994 vol. 269 p. 24107 - 24113

0021-9258

Chemical modification studies of spinach leaf ADPglucosepyrophosphorylase with pyridoxal-P haveshown that a highly conserved lysyl residue near the Cterminus might be involvedin theb inding of 3-P-glycerate,the allosteric activator. Site-directed mutagenesis ofthe corresponding residue (Lys419) of the Anabaena enzymewas performed to determine the role of this conservedresidue. Replacing Lys419 with either arginine,alanine, glutamine, or glutamic acid produced mutantenzymes with apparent affinities for 3-P-glycerate, 25-150-fold lower than that of the wild-type enzyme. Themutant enzymes, however, werset ill activated to a greatextent at higher concentrations of activator suggestingthat an additional site or residue was involved in thebinding of the activator. These mutations caused lesseror no effect on the kinetic constants for the substratesand inhibitor, Pi, as well as on the catalytic efficiencyand thermal stability. The results suggest that both thecharge and size of lysine residue 419 are required for theproper binding of 3-P-glycerate. Chemical modificationof the Anabaena wild-type enzyme with pyridoxal-iPn -dicated that Lys419 was the only lysyl residue modified.We further performed the same experiment on theK419R mutant enzyme and found another lysyl residue,LysS8’, was modified. Reductive phosphopyridoxylationof the wild-type and K419R enzymes caused dramaticalteration in allosteric properties. The activator, 3-Pglycerate,and inhibitor, Pi, protected the enzyme fromreductive phosphopyridoxylation. The modified enzymeswere more active in the absence of 3-P-glycerateand less sensitive to Pi inhibition.