Glutathione is a thiol-containing tripeptide that plays important roles in redox-related processes. The first step in glutathione biosynthesis is catalysed by γ-GCS (γ-glutamylcysteine synthetase). The crystal structure of Escherichia coli γ-GCS has revealed the presence of a disulfide bond. As the disulfide-bonding cysteine residues Cys372 and Cys395 are not well conserved among γ-GCS enzymes in this lineage, we have initiated a biochemical genetic strategy to investigate the functional importance of these and other cysteine residues. In a cysteine-free γ-GCS that was non-functional, suppressor analysis yielded combinations of cysteine and aromatic residues at the position of the disulfide bond, and one mutant that lacked any cysteine residues. Kinetic analysis of the wild-type and mutant enzymes revealed that the disulfide bond was not involved in determining the affinity of the enzyme towards its substrate, but had an important role in determining the stability of the protein, and its catalytic efficiency. We show that in vivo the γ-GCS enzyme can also exist in a reduced form and that the mutants lacking the disulfide bond show a decreased half-life. These results demonstrate a novel means of regulation of γ-GCS by the redox environment that works by an alteration in its stability.
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Research Article|
January 09 2013
Redox-dependent stability of the γ-glutamylcysteine synthetase enzyme of Escherichia coli: a novel means of redox regulation
Shailesh Kumar;
Shailesh Kumar
1Institute of Microbial Technology (CSIR), Sector 39-A, Chandigarh 160 036, India
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Neha Kasturia;
Neha Kasturia
1
1Institute of Microbial Technology (CSIR), Sector 39-A, Chandigarh 160 036, India
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Amit Sharma;
Amit Sharma
2
1Institute of Microbial Technology (CSIR), Sector 39-A, Chandigarh 160 036, India
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Manish Datt;
Manish Datt
3
1Institute of Microbial Technology (CSIR), Sector 39-A, Chandigarh 160 036, India
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Anand K. Bachhawat
Anand K. Bachhawat
4
1Institute of Microbial Technology (CSIR), Sector 39-A, Chandigarh 160 036, India
4To whom correspondence should be addressed at the present address: Indian Institute of Science Education and Research, Mohali, S.A.S. Nagar, Punjab 140306, India (email anand@iisermohali.ac.in).
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Publisher: Portland Press Ltd
Received:
February 02 2012
Revision Received:
October 29 2012
Accepted:
November 05 2012
Accepted Manuscript online:
November 05 2012
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2013 Biochemical Society
2013
Biochem J (2013) 449 (3): 783–794.
Article history
Received:
February 02 2012
Revision Received:
October 29 2012
Accepted:
November 05 2012
Accepted Manuscript online:
November 05 2012
Citation
Shailesh Kumar, Neha Kasturia, Amit Sharma, Manish Datt, Anand K. Bachhawat; Redox-dependent stability of the γ-glutamylcysteine synthetase enzyme of Escherichia coli: a novel means of redox regulation. Biochem J 1 February 2013; 449 (3): 783–794. doi: https://doi.org/10.1042/BJ20120204
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