Properties of the Bacillus licheniformis A5 glutamine synthetase purified from cells grown in the presence of ammonia or nitrate.
The glutamine synthetase from Bacillus licheniformis A5 was purified by using a combination of polyethylene glycol precipitation and chromatography on Bio-Gel A 1.5m. The resulting preparation was judged to be homogeneous by the criteria of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, equilibrium analytical ultracentrifugation, and electron microscopic analysis. The enzyme is a dodecamer with a molecular weight of approximately 616,000, and its subunit molecular weight is 51,000. Under optimal assay conditions (pH 6.6, 37 degrees C) apparent Km values for glutamate, ammonia, and manganese.adenosine 5'-triphosphate (1:1 ratio) were 3.6, 0.4, and 0.9 mM, respectively. Glutamine synthetase activity was inhibited approximately 50% by the addition of 5 mM glutamine, alanine, glycine, serine, alpha-ketoglutarate, carbamyl phosphate, adenosine 5'-diphosphate, or inosine 5'-triphosphate to the standard glutamine synthetase assay system, whereas 5 mM adenosine 5'-monophosphate or pyrophosphate caused approximately 90% inhibition of enzyme activity. Phosphorylribosyl pyrophosphate at 5 mM enhanced activity approximately 60%. We were unable to detect any physical or kinetic differences in the properties of the enzyme when it was purified from cells grown in the presence of ammonia or nitrate as sole nitrogen source. The data indicate that B. licheniformis A5 contains one species of glutamine synthetase whose catalytic activity is not regulated by a covalent modification system.
D000596:Amino Acids / Q000494:pharmacology; D000645:Ammonium Sulfate / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology*; D002413:Cations, Divalent / Q000494:pharmacology; D011756:Diphosphates / Q000494:pharmacology; D005974:Glutamate-Ammonia Ligase / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009566:Nitrates / Q000378:metabolism*; D009711:Nucleotides / Q000494:pharmacology; D017680:Potassium Compounds
D000596:Amino Acids; D002413:Cations, Divalent; D011756:Diphosphates; D009566:Nitrates; D009711:Nucleotides; D017680:Potassium Compounds; D005974:Glutamate-Ammonia Ligase; C023844:potassium nitrate; D000645:Ammonium Sulfate