Biosynthesis of bacterial glycogen. Activator-induced oligomerization of a mutant Escherichia coli ADP-glucose synthase.
ADP-glucose synthase (EC 2.7.7.27) was purified to homogeneity from SG5-504, Aa mutant strain of Escherichia coli B. It had a molecular weight of approximately 2 X 10(5) and four identical subunits. In the presence of its allosteric activator, fructose-1-6-P2, the mutant enzyme formed oligomers with several times the tetramer molecular weight, as revealed by sedimentation equilibrium centrifugation, polyacrylamide gel electrophoresis, and gel filtration column chromatography. Enzyme purified from a wild type strain, AC70R1, did not exhibit the activator-induced oligomerization. Catalytically, the mutant enzyme had a 2-fold higher affinity for activator and a 2- to 3-fold lower affinity for the allosteric inhibitors 5'-adenylate and KH2PO4 than the wild type. The two enzymes appeared identical in subunit molecular weight, amino acid composition, COOH-terminal amino acid, and the first 27 residues of the NH2-terminal sequence. Subtle structural differences were revealed by chemical modification of specific residues. Compared to wild type, the mutant enzyme had 1 cysteine/subunit more accessible to modification by Ellman's reagent. SG5-504 enzyme was inactivated more slowly than AC70R1 enzyme by phenylglyoxal and by trinitrobenzenesulfonate. These results reflected an altered conformation of the SG5-504 enzyme molecule. The unique ability of it to oligomerize in the presence of activator may be the result of a single amino acid substitution.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002268:Carboxypeptidases; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D005636:Fructosephosphates / Q000494:pharmacology; D005958:Glucosephosphates; D006003:Glycogen / Q000096:biosynthesis*; D006598:Hexosediphosphates / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009154:Mutation; D009713:Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D011725:Pyridines / Q000494:pharmacology
D000596:Amino Acids; D005636:Fructosephosphates; D005958:Glucosephosphates; D006598:Hexosediphosphates; D046911:Macromolecular Substances; D011725:Pyridines; D006003:Glycogen; D004228:Dithionitrobenzoic Acid; D009713:Nucleotidyltransferases; D002268:Carboxypeptidases