GLUTAMINE SYNTHETASE DEADENYLYLATING ENZYME SYSTEM FROM ESCHERICHIA COLI . RESOLUTION INTO 2 COMPONENTS SPECIFIC NUCLEOTIDE STIMULATION AND COFACTOR REQUIREMENTS

Glutamine synthetase from Escherichia coli exists as a dodecameric aggregate which has varying amounts of 5′-adenylyl groups bound to it; there may be from 0 to 12 equiv of adenylyl groups bound per mole of enzyme. The adenylyl residues are hydrolyzed from glutamine synthetase in a reaction catalyzed by the glutamine synthetase deadenylylating enzyme, which has been purified 20-fold by a combination of protamine sulfate fractionation, ammonium sulfate fractionation, and adsorption to and elution from alumina C-γ gel. The enzyme may be resolved into two components, both of which are required for activity, by gel filtration on Bio- Gel A-0.5, in the presence of phosphate buffer and dithiothreitol. The enzyme The enzyme requires either Mn2+ or Mg2+ for activity, with the former being effective at one-tenth the concentration of the latter. Ca2+, Cd2+, Zn2+, and Ba2+ are not activators of the reaction. The pH optimum for deadenylylation is rather sharp at pH 7.3. Deadenylylating enzyme activity is stimulated more than twofold by inorganic phosphate or arsenate; sulfate has little effect on the reaction, and pyrophosphate completely inhibits it. The deadenylylation reaction is stimulated considerably by specific nucleotides. Thus, more than 45-fold activation is afforded by a combination of uridine triphosphate (0.8 mM) and adenosine triphosphate (40μM). Adenosine triphosphate is most effective when added to subsaturating concentrations of uridine triphosphate; under these conditions, uridine triphosphate and adenosine triphosphate behave in a synergistic fashion in stimulating deadenylylation. A ribosomal ribonucleic acid preparation from E. coli may replace adenosine triphosphate in the synergistic stimulation of the reaction, as may adenosine diphosphate. A precursor of glutamine, -ketoglutarate, stimulates the deadenylylation reaction 15-fold; glutamine itself causes almost complete inhibition. The concentration of α-ketoglutarate which effects half-maximal activation is 1.0 mM. The concentration of glutamine which inhibits the reaction 50% is 0.25 mM. A model is presented which suggests that the relative concentrations of α-ketoglutarate, glutamate, glutamine, and the products of glutamine metabolism are the decisive factors in the control of nitrogen metabolism in E. coli. © 1969, American Chemical Society. All rights reserved.