Journal
PLANT SCIENCE
Volume 154, Issue 2, Pages 189-197Publisher
ELSEVIER SCI IRELAND LTD
DOI: 10.1016/S0168-9452(00)00197-7
Keywords
glutamine synthetase; plant nitrogen assimilation; structure-function; site-directed mutagenesis; recombinant enzymes; Phaseolus vulgaris
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The residue Cys-92 from the alpha-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. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.
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