In this paper we examine the functionality of Glu-297 from the alpha-polypeptide of Phaseolus vulgaris glutamine synthetase (EC 6.3.1.2). For this purpose, the gln alpha cDNA was recombinantly expressed in Escherichia coli, and site-directed mutants constructed, in which this residue was replaced by alanine. The level of glutamine synthetase transferase catalytic activity in the mutant strain was 70-fold lower while biosynthetic activity remained practically unaffected. Kinetic parameters for both enzyme activities were not greatly altered except for the Km for ammonium in biosynthetic activity, which increased 100-fold. A similar result was reported when mutagenizing Glu-327 from E. coli glutamine synthetase, a residue shown to be present at the active site. This suggests that the Glu residue mutated in the higher-plant enzyme could develop a similar catalytic role to that of bacteria. Another characteristic feature of the mutant protein was its higher resistance to inhibition of the biosynthetic activity by L-methionine sulfoximine, a typical inhibitor of glutamine synthetase. In addition, we show that immunoreactivity of the glutamine synthetase mutant protein, both under native and denaturing conditions, is similar to the wild type, indicating that no deep conformational changes were produced as a consequence of the introduced mutation. However, structural changes in the active site can be predicted from alterations detected in the behaviour of the mutant protein towards affinity chromatography on 2',5'-ADP-Sepharose, as compared to the wild type. Nevertheless, complementation of an E. coli glnA mutation indicated that the E297A mutant enzyme was physiologically functional.