Redox-dependent stability of the γ-glutamylcysteine synthetase enzyme of Escherichia coli: a novel means of redox regulation

Glutathione is a thiol-containing tripeptide that plays important roles in redox-related processes. The first step in glutathione biosynthesis is catalysed by gamma-GCS (gamma-glutamylcysteine synthetase). The crystal structure of Escherichia coli gamma-GCS has revealed the presence of a disulfide bond. As the disulfide-bonding cysteine residues Cys(372) and Cys(395) are not well conserved among gamma-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 gamma-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 gamma-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 gamma-GCS by the redox environment that works by an alteration in its stability.