Identification of an essential cysteinyl residue for the structure of glutamine synthetase α from Phaseolus vulgaris
| Autor: | Marco Betti, Pilar Guardado, Raymond Buser, Guillermo Estivill, Antonio Joaquín Santos Márquez |
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| Rok vydání: | 2010 |
| Předmět: |
Time Factors
Molecular Sequence Data Plant Science Biology Protein Structure Secondary Structure-Activity Relationship Adenosine Triphosphate Glutamate-Ammonia Ligase Sequence Analysis Protein Glutamine synthetase Escherichia coli Genetics Amino Acid Sequence Cysteine Phaseolus Alanine chemistry.chemical_classification Sulfhydryl Reagents Recombinant Proteins Enzyme structure Enzyme assay Enzyme Activation Glutamine Spectrometry Fluorescence Enzyme chemistry Biochemistry Biocatalysis Mutagenesis Site-Directed biology.protein Electrophoresis Polyacrylamide Gel Mutant Proteins Protein quaternary structure Sequence Alignment |
| Zdroj: | Planta. 231:1101-1111 |
| ISSN: | 1432-2048 0032-0935 |
| DOI: | 10.1007/s00425-010-1115-z |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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