title: Biosynthesis of bacterial glycogen. Determination of the amino acid changes that alter the regulatory properties of a mutant Escherichia coli ADP-glucose synthetase.
abstract: The ADP-glucose synthetase of Escherichia coli K12 mutant 618 has a higher apparent affinity for the activator, fructose 1,6-P2 and a lower apparent affinity for the inhibitor, 5'-AMP, than the normal enzyme. The structural gene, glgC, of the mutant enzyme has been cloned and sequenced (Lee, Y. M., Kumar, A., and Preiss, J. (1987) Nucleic Acids Res. 15, 10603). Substitutions in the mutant enzyme were amino acid residues 296 (Lys to Glu) and 336 (Gly to Asp). Single mutant enzymes, Glu296 and Asp336, were constructed using oligonucleotide-directed mutagenesis. The Glu296 enzyme had the same allosteric kinetic constants as the wild type enzyme. The Asp336 enzyme was catalytically defective. Thus, the mutations at 296 and at 336 separately could not account for the allosteric alterations of the mutant enzyme. A hybrid glgC gene was prepared from genes of wild type and mutant 618 glgC using DNA recombinant techniques. The C-terminal portion of mutant 618 containing Glu296 and Asp336, combined with the N-terminal portion of wild type enzyme, showed allosteric and substrate kinetics similar to mutant 618 enzyme. Thus, alteration of the normal allosteric properties in mutant 618 are due to changes of both Lys296 to Glu and Gly336 to Asp.
authors.affiliation: Department of Biochemistry, Michigan State University, East Lansing 48824.
authors.forename: A
authors.initials: A
authors.lastname: Kumar
authors.forename: P
authors.initials: P
authors.lastname: Ghosh
authors.forename: Y M
authors.initials: YM
authors.lastname: Lee
authors.forename: M A
authors.initials: MA
authors.lastname: Hill
authors.forename: J
authors.initials: J
authors.lastname: Preiss
mesh_terms: D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D005822:Genetic Vectors; D051599:Glucose-1-Phosphate Adenylyltransferase; D006003:Glycogen / Q000096:biosynthesis*; D007700:Kinetics; D009154:Mutation; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
chemical_list: D006003:Glycogen; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase