636-58-8Relevant articles and documents
Structural basis for feedback and pharmacological inhibition of Saccharomyces cerevisiae glutamate cysteine ligase
Biterova, Ekaterina I.,Barycki, Joseph J.
, p. 14459 - 14466 (2010)
Structural characterization of glutamate cysteine ligase (GCL), the enzyme that catalyzes the initial, rate-limiting step in glutathione biosynthesis, has revealed many of the molecular details of substrate recognition. To further delineate the mechanistic details of this critical enzyme, we have determined the structures of two inhibited forms of Saccharomyces cerevisiae GCL (ScGCL), which shares significant sequence identity with the human enzyme. In vivo, GCL activity is feedback regulated by glutathione. Examination of the structure of ScGCL-glutathione complex (2.5 A ; R = 19.9%, Rfree = 25.1%) indicates that the inhibitor occupies both the glutamate- and the presumed cysteine-binding site and disrupts the previously observed Mg2+ coordination in the ATP-binding site. L-Buthionine-S-sulfoximine (BSO) is a mechanism-based inhibitor of GCL and has been used extensively to deplete glutathione in cell culture and in vivo model systems. Inspection of the ScGCL-BSO structure (2.2 A ; R = 18.1%, Rfree = 23.9%) confirms that BSO is phosphorylated on the sulfoximine nitrogen to generate the inhibitory species and reveals contacts that likely contribute to transition state stabilization. Overall, these structures advance our understanding of the molecular regulation of this critical enzyme and provide additional details of the catalytic mechanism of the enzyme.
Redox-dependent stability of the γ-glutamylcysteine synthetase enzyme of Escherichia coli: A novel means of redox regulation
Kumar, Shailesh,Kasturia, Neha,Sharma, Amit,Datt, Manish,Bachhawat, Anand K.
, p. 783 - 794 (2013)
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 showthat 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. The Authors Journal compilation
Synthesis of hydroxymethylglutathione from glutathione and L-serine catalyzed by carboxypeptidase Y.
Okumura, Ryosuke,Koizumi, Yukio,Sekiya, Jiro
, p. 434 - 437 (2007/10/03)
Hydroxymethylglutathione (gamma-L-glutamyl-L-cysteinyl-L-serine; hmGSH) occurs in many species belonging to the family Gramineae, but the biosynthetic pathway for hmGSH has not been identified. We found that carboxypeptidase Y (CPY), but not carboxypeptidase A, catalyzed hmGSH synthesis from glutathione and L-serine in vitro at acidic pH. CPY also catalyzed methylglutathione synthesis from glutathione and L-alanine. These findings suggested that a carboxypeptidase-like enzyme may be involved in hmGSH synthesis in vivo.