In the course of studies on glutaminedependent carbamyl phosphate synthetase from Aerobacter aerogenes, we purified another protein which was found to be glutamate synthase (EC 2.6.1.53). The enzyme, obtained in apparently homogeneous form (monomer molecular weight about 227,000; s2ow = 17.6 S), was found to be a typical glutamine amidotransferase in that it exhibits glutaminase activity and can utilize ammonia in place of glutamine as a nitrogen donor. Earlier work in this laboratory showed that glutamine-dependent carbamyl phosphate synthetase from Escherichia coli consists of a heavy subunit which can catalyze the synthesis of carbamyl phosphate from ATP, bicarbonate, and ammonia (but not from glutamine), and a light subunit which functions to bind glutamine (1, 2). In subsequent hybridization experiments with glutamine dependent carbamyl phosphate synthetase from Aerobacter aerogenes, it was found that the light subunit of the A. aerogenes enzyme could combine with the heavy subunit of the E. coli enzyme to form an active glutamine-dependent hybrid enzyme; similarly, an active hybrid enzyme could be prepared from the light subunit of the E. coli enzyme and the heavy subunit of the A. aerogenes enzyme.* In the course of isolating glutamine-dependent carbamyl phosphate synthetase from A. aerogenes, we noted during a gel filtration step in the procedure that another protein had also been purified (see first peak, Fig. 1). The catalytic properties of this protein were initially obscure, but we decided to pursue study of this apparently homogeneous protein after we found that it exhibited glutaminase activity and that it could be split to a heavy and a light subunit when subjected to polyacrylamide gel electrophoresis in sodium dodecyl sulfate.These observations led us to think that the new protein might be a glutanmine amidotransferase, and after testing it for the activities of the several known enzymes in this category we discovered that the new protein is glutamate synthase (EC 2.6.1.53) whose major catalytic activity is to form glutamate from a-ketoglutarate and glutamine according to the following reaction (3-5): a-ketoglutarate + TPNH + H+ + L-glutamine 2 L-glutamate + TPN+ Several glutamine amidotransferases [e.g., carbamyl phosphate synthetase (1, 2), anthranilate synthetase (6), p-aminobenzoate synthetase (7) ] are composed of a heavy and a light subunit, and there is strong evidence in each case that the light subunit has the function of binding glutarmine; the amide nitrogen is then transferred to the heavy subunit for use in the corresponding synthesis reactions. We therefore expected that the light subunit of A. aerogenes glutamate synthase would contain the glutamine binding site of this enzyme. However, as described here, we found that the glutamine binding site of glutamate synthase is located on the heavy subunit. Miller and Stadtman (8,9) had previously shown that glutamate synthase from E. coli is an iron-sulfide flavoprotein. We have found that the A. aerogenes glutamate synthase is a...