title: Arginine294 is essential for the inhibition of Anabaena PCC 7120 ADP-glucose pyrophosphorylase by phosphate.
abstract: Treatment of ADP-glucose pyrophosphorylase (EC 2.7.7.27) from the cyanobacterium Anabaena PCC 7120 with phenylglyoxal in 50 mM Hepes, pH 8.0, at 25 degrees C resulted in a time- and concentration-dependent loss of enzyme activity. Phosphate, the inhibitor, protected the enzyme from inactivation most effectively, while 3-P-glycerate, fructose-1,6-P2, pyridoxal-P, and ATP plus magnesium were also good protectors. After incubation with 2 mM phenylglyoxal for 1 h, the modified enzyme had a 10-fold lower apparent affinity for phosphate in the absence of the activator, 3-P-glycerate, than that of the wild-type enzyme. This result has implicated the involvement of an arginine residue at the allosteric sites, most probably the inhibitor-binding site, of ADP-glucose pyrophosphorylase from the cyanobacterium Anabaena PCC 7120. In order to identify the arginine residue, five arginine residues, which are conserved in all higher-plant and cyanobacterial enzymes but not in enteric bacterial enzymes, were individually converted to alanine by site-directed mutagenesis. The mutant enzymes, R66A, R105A, R294A, and R385A, were purified, and the properties of these mutants were compared with the wild-type enzyme. Substitution of arginine294 with alanine resulted in an enzyme with more than 100-fold or 40-fold lower affinity for the inhibitor, phosphate, in the absence or presence of 3-P-glycerate, respectively. This mutation had no or lesser impact on the kinetic constants for the substrates and the activator, 3-P-glycerate.
authors.affiliation: Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, USA.
authors.forename: J
authors.initials: J
authors.lastname: Sheng
authors.forename: J
authors.initials: J
authors.lastname: Preiss
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000409:Alanine; D019943:Amino Acid Substitution; D017033:Anabaena / Q000201:enzymology*; D001120:Arginine; D001665:Binding Sites; D005635:Fructosediphosphates / Q000494:pharmacology; D051599:Glucose-1-Phosphate Adenylyltransferase; D005988:Glyceric Acids / Q000494:pharmacology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D010658:Phenylglyoxal / Q000494:pharmacology*; D010710:Phosphates / Q000494:pharmacology*; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism
chemical_list: D005635:Fructosediphosphates; D005988:Glyceric Acids; D010710:Phosphates; D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; C005156:3-phosphoglycerate; D000255:Adenosine Triphosphate; D001120:Arginine; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase; C029063:fructose-1,6-diphosphate; D010658:Phenylglyoxal; D000409:Alanine