Function of conserved residues of human glutathione synthetase: implications for the ATP-grasp enzymes.
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.
Departments of Chemistry and Biology, University of North Texas, Denton, Texas 76203, USA.
Adriana
A
Dinescu
Thomas R
TR
Cundari
Vikas S
VS
Bhansali
Jia-Li
JL
Luo
Mary E
ME
Anderson
D000255:Adenosine Triphosphate / Q000737:chemistry*; D001216:Asparagine / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry; D005981:Glutathione Synthase / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008239:Lysine / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012984:Software
D008024:Ligands; D011994:Recombinant Proteins; D018698:Glutamic Acid; D001216:Asparagine; D000255:Adenosine Triphosphate; D004247:DNA; D005981:Glutathione Synthase; D005978:Glutathione; D008239:Lysine