PURIFICATION AND PARTIAL CHARACTERIZATION OF GLUTAMATE SYNTHASE FROM RHODOSPIRILLUM-RUBRUM GROWN UNDER NITROGEN-FIXING CONDITIONS

Glutamate synthase, a key enzyme in ammonia assimilation, has been purified from the photosynthetic bacterium Rhodospirillum rubrum. The purification procedure involves ion-exchange chromatography, affinity chromatography and gel filtration. The recovery in the procedure is high (62 %) and the specific activity is 21-mu-mol of NADPH oxidized/min per mg. The enzyme is specific for its substrates, and no activity was demonstrated with NADH or NH4+ ions substituting for NADPH and glutamine respectively. The enzyme is composed of two dissimilar subunits with molecular masses of 53 and 152 kDa, and it is shown that Cl- ions have an effect on the aggregation of the enzyme. K(m) values for the substrates are: NADPH, 16-mu-M; 2-oxoglutarate, 10-mu-M; and glutamine, 65-mu-M. The enzyme is inhibited by amidotransferase inhibitors at micromolar concentrations. The role of the enzyme in the metabolic regulation of nitrogenase is discussed.