Site-directed mutagenesis of Cys-92 from the α-polypeptide of Phaseolus vulgaris glutamine synthetase reveals that this highly conserved residue is not essential for enzyme activity but it is involved in thermal stability

Abstract

The residue Cys-92 from the α-polypeptide of Phaseolus vulgaris glutamine synthetase is a highly conserved residue in prokaryotic and eukaryotic glutamine synthetase genes. This cysteine residue was previously proposed as a good candidate for being essential for enzyme activity. We have examined through heterologous expression in Escherichia coli and site-directed mutagenesis the functional importance of this residue. We have found that the thiol group of Cys-92 is not essential either for glutamine synthetase biosynthetic or transferase enzyme activities. The characteristic inhibition by p-hydroxymercuribenzoate (a specific sulphydryl reagent) was not substantially altered as a consequence of replacement of Cys-92 by Ala. Immunoreactivity of the glutamine synthetase mutant protein, examined both under native and denaturing conditions, was similar to the wild-type, indicating that no significant conformational changes were produced as a consequence of the introduced mutation. However, the mutant enzyme C92A was considerably less stable than the wild-type. These results indicate that Cys-92 is not an essential residue for enzyme activity but it is important for stability of the glutamine synthetase protein.