Functional importance of Asp56 from the α-polypeptide of Phaseolus vulgaris glutamine synthetase -: An essential residue for transferase but not for biosynthetic enzyme activity

Replacement of Asp56 by site-directed mutagenesis of the a-gene from Phaseolus vulgaris glutamine synthetase heterologously expressed in Escherichia coli produces a complete loss of transferase enzyme activity, thus revealing essentiality of the residue for this particular enzyme activity. This happens independent of Asp56 being replaced by Ala or Glu, suggesting that the essentiality of this residue cannot be attributed to its negative electrical charge. However, a high level of glutamine synthetase biosynthetic specific activity (referred to glutamine synthetase protein, as determined immunologically), is present in D56A and D56E mutants, suggesting that Asp56 is an example of a residue that has a different role in the catalytic mechanism of both enzyme activities of this protein. K-m for ATP, glutamate and Mg2+, as well as energy of activation, can be altered as a consequence of the performed mutations. However, the K-m and catalytic efficiency for ammonium remains unaffected. Therefore, the catalytic role of Asp56 in the alpha-polypeptide of higher plant glutamine synthetase is quite different from the role proposed for its highly conserved homologue in bacteria (Asp50 in E. coli), which has been associated with binding and deprotonation of ammonium. On the other hand, we also show other results indicating that Asp56 is important in the spatial conformation of the active site and/or the protein, Asp56 being a crucial residue in the salting-out aggregation properties of the enzyme.