Abstract
Glutathione synthetase is an enzyme that belongs to the glutathione synthetase ATP-binding domain-like superfamily. It catalyzes the second step in the biosynthesis of glutathione from gamma-glutamylcysteine and glycine in an ATP-dependent manner. Glutathione synthetase has been purified and sequenced from a variety of biological sources; still, its exact mechanism is not fully understood. A variety of structural alignment methods were applied and four highly conserved residues of human glutathione synthetase (Glu-144, Asn-146, Lys-305, and Lys-364) were identified in the binding site. The function of these was studied by experimental and computational site-directed mutagenesis. The three-dimensional coordinates for several human glutathione synthetase mutant enzymes were obtained using molecular mechanics and molecular dynamics simulation techniques, starting from the reported crystal structure of human glutathione synthetase. Consistent with circular dichroism spectroscopy, our results showed no major changes to overall enzyme structure upon residue mutation. However, semiempirical calculations revealed that ligand binding is affected by these mutations. The key interactions between conserved residues and ligands were detected and found to be essential for enzymatic activity. Particularly, the negatively charged Glu-144 residue plays a major role in catalysis.
Highlights
Glutathione synthetase is an enzyme that belongs to (␥-glutamylcysteinyl phosphate), followed by attack of the glythe glutathione synthetase ATP-binding domain-like su- cine and formation of an enzyme-product complex, which fiperfamily
Similarities between Glutathione Synthetase and Members of ATP-grasp Superfamily—Structural Classification of Proteins (SCOP) [45] analysis shows that human glutathione synthetase (PDB code ϭ 2HGS) belongs to the Pre-ATP-grasp superfamily, which is the only member of the Pre-ATP-grasp fold
The current work is the first study that examines all members of the Pre-ATP-grasp superfamily, FIG. 4—continued including human glutathione synthetase, on the subject of highly conserved residues and their function
Summary
Glutathione synthetase is an enzyme that belongs to (␥-glutamylcysteinyl phosphate), followed by attack of the glythe glutathione synthetase ATP-binding domain-like su- cine and formation of an enzyme-product complex, which fiperfamily. A variety of structural alignment methods were applied and four highly conserved residues of human glutathione synthetase (Glu-144, Asn-146, Lys-305, and Lys-364) were identified in the binding site. On the basis of clinical step in the biosynthesis of glutathione (GSH) from ␥-glutamyl- symptoms, patients with glutathione synthetase deficiency can cysteine and glycine in an ATP-dependent manner. This proc- be classified into three phenotypes: mild, moderate, and severe ess involves formation of an enzyme-bound acyl phosphate (or generalized).
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