title: Redox-dependent stability of the γ-glutamylcysteine synthetase enzyme of Escherichia coli: a novel means of redox regulation.
abstract: 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.
authors.affiliation: Institute of Microbial Technology (CSIR), Sector 39-A, Chandigarh 160 036, India.
authors.forename: Shailesh
authors.initials: S
authors.lastname: Kumar
authors.forename: Neha
authors.initials: N
authors.lastname: Kasturia
authors.forename: Amit
authors.initials: A
authors.lastname: Sharma
authors.forename: Manish
authors.initials: M
authors.lastname: Datt
authors.forename: Anand K
authors.initials: AK
authors.lastname: Bachhawat
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005721:Glutamate-Cysteine Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D005721:Glutamate-Cysteine Ligase; D003545:Cysteine