Human glutathione synthetase (hGS) catalyzes the second ATP-dependent step in the biosynthesis of glutathione (GSH) and is negatively cooperative to the gamma-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 Km 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. (C) 2011 Elsevier Inc. All rights reserved.