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Article Dans Une Revue Biochemical and Biophysical Research Communications Année : 2011

Aspartate 458 of human glutathione synthetase is important for cooperativity and active site structure

Teresa Brown
  • Fonction : Auteur
Michael Drummond
  • Fonction : Auteur
Sarah Barelier
Amanda Crutchfield
  • Fonction : Auteur
Adriana Dinescu
  • Fonction : Auteur
Kerri Slavens
  • Fonction : Auteur
Thomas Cundari
  • Fonction : Auteur
Mary Anderson
  • Fonction : Auteur

Résumé

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.

Dates et versions

hal-03766722 , version 1 (01-09-2022)

Identifiants

Citer

Teresa Brown, Michael Drummond, Sarah Barelier, Amanda Crutchfield, Adriana Dinescu, et al.. Aspartate 458 of human glutathione synthetase is important for cooperativity and active site structure. Biochemical and Biophysical Research Communications, 2011, 411 (3), pp.536-542. ⟨10.1016/j.bbrc.2011.06.166⟩. ⟨hal-03766722⟩
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