title: Aspartate 458 of human glutathione synthetase is important for cooperativity and active site structure.
abstract: Human glutathione synthetase (hGS) catalyzes the second ATP-dependent step in the biosynthesis of glutathione (GSH) and is negatively cooperative to the γ-glutamyl substrate. The hGS active site is composed of three highly conserved catalytic loops, notably the alanine rich A-loop. Experimental and computational investigations of the impact of mutation of Asp458 are reported, and thus the role of this A-loop residue on hGS structure, activity, negativity cooperativity and stability is defined. Several Asp458 hGS mutants (D458A, D458N and D458R) were constructed using site-directed mutagenesis and their activities determined (10%, 15% and 7% of wild-type hGS, respectively). The Michaelis-Menten constant (K(m)) was determined for all three substrates (glycine, GAB and ATP): glycine K(m) increased by 30-115-fold, GAB K(m) decreased by 8-17-fold, and the ATP K(m) was unchanged. All Asp458 mutants display a change in cooperativity from negative cooperativity to non-cooperative. All mutants show similar stability as compared to wild-type hGS, as determined by differential scanning calorimetry. The findings indicate that Asp458 is essential for hGS catalysis and that it impacts the allostery of hGS.
authors.affiliation: Department of Chemistry and Physics, Texas Woman's University, Denton, TX 76204, United States.
authors.forename: Teresa R
authors.initials: TR
authors.lastname: Brown
authors.forename: Michael L
authors.initials: ML
authors.lastname: Drummond
authors.forename: Sarah
authors.initials: S
authors.lastname: Barelier
authors.forename: Amanda S
authors.initials: AS
authors.lastname: Crutchfield
authors.forename: Adriana
authors.initials: A
authors.lastname: Dinescu
authors.forename: Kerri D
authors.initials: KD
authors.lastname: Slavens
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Cundari
authors.forename: Mary E
authors.initials: ME
authors.lastname: Anderson
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain; D005981:Glutathione Synthase / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary
chemical_list: D001224:Aspartic Acid; D005981:Glutathione Synthase