The glutamine synthetase from Azotobacter vinelandii: purification, characterization, regulation and localization.
The glutamine synthetase (EC 6.3.1.2) from the N2-fixing bacterium Azotobacter vinelandii was purified to homogeneity by heat treatment, ammonium sulfate precipitation and ion-exchange chromatography. The following molecular parameters were determined: molecular weight 640 000, subunit molecular weight 53 000, partial specific volume 0.710 cm3/g, isoelectric point 4.6, amino acid composition. Most of the molecules are composed of 12 identical subunits but active oligomers of other degrees of polymerization, apparently aggregates with 8, 10 and 24 subunits, were also detected to a lesser extent. The enzymatic activity is regulated via adenylylation-deadenylylation cycles: liberation of AMP was detected upon treatment of the adenylylated form with phosphodiesterase along with a change in the catalytic properties. Adenylylation in vivo is specifically induced by high extracellular ammonia levels. The Km values for the Mg2+-dependent formation of glutamine were independent of the degree of adenylylation for glutamate and ATP, but varied for ammonia. Furthermore the catalytic activity is regulated by several nitrogenous feedback inhibitors. The degree of inhibition in some cases was dependent on the substrate concentrations: the sensitivity towards glycine, alanine and serine decreased with a decreasing ammonia level, while the sensitivity towards ADP or AMP increased with a decreasing ATP concentration. Part of the enzyme (about 30%) seems to be attached to the plasma membrane while the main fraction is found in the cytosol.
D000596:Amino Acids / Q000032:analysis; D000641:Ammonia / Q000494:pharmacology; D001395:Azotobacter / Q000187:drug effects / Q000201:enzymology*; D005974:Glutamate-Ammonia Ligase / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight
D000596:Amino Acids; D046911:Macromolecular Substances; D008345:Manganese; D000641:Ammonia; D005974:Glutamate-Ammonia Ligase