Identification of an essential cysteinyl residue for the structure of glutamine synthetase alpha from Phaseolus vulgaris.
We have studied the possible role, in a plant glutamine synthetase (GS), of the different cysteinyl residues present in this enzyme. For this purpose we carried out the site-directed mutagenesis of the cDNA for alpha-GS polypeptide from Phaseolus vulgaris in the positions corresponding to Cys-92, Cys-159, and Cys-179, followed by heterologous expression in E. coli and enzymatic characterisation of WT and mutant proteins. The results show that neither Cys-92 nor Cys-179 residues were essential for enzyme activity, but the replacement of Cys-159 by alanine or serine strongly affects the quaternary structure and function of the GS enzyme molecule, resulting in a complete loss of enzymatic activity. Other studies using sulfhydryl specific reagents such as pHMB (p-hydroxymercuribenzoate) or DTNB (5,5'-dithiobis-2-nitrobenzoate) confirmed that the profound inhibition produced is associated with an important alteration of the quaternary structure of GS, and suggest that Cys-159 might be the residue responsible for the enzyme inhibition. All these results suggest that the Cys-159 residue is essential for the enzyme structure. The results are also consistent with previous reports based on classical biochemistry studies indicating the presence of essential cysteinyl residues for the enzyme activity of higher plant GS.
Departamento de Bioquímica Vegetal y Biología Molecular, Facultad de Química, Universidad Sevilla, Seville, Spain.
Guillermo
G
Estivill
Pilar
P
Guardado
Raymond
R
Buser
Marco
M
Betti
Antonio J
AJ
Márquez
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D055162:Biocatalysis / Q000187:drug effects; D003545:Cysteine / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000302:isolation & purification / Q000378:metabolism; D027805:Phaseolus / Q000187:drug effects / Q000201:enzymology*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013997:Time Factors
D050505:Mutant Proteins; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D000255:Adenosine Triphosphate; D005974:Glutamate-Ammonia Ligase; D003545:Cysteine