Date: August 5, 2011
Creator: Brown, Teresa R.; Drummond, Michael L.; Barelier, Sarah; Crutchfield, Amanda S.; Dinescu, Adriana; Slavens, Kerri D. et al.
Description: Article discussing ASPARTATE 458 of human glutathione synthetase. 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, D458R) were constructed using site-directed mutagenesis and their activities determined (10, 15, and 7% of wild-type hGS, respectively). The Michaelis-Menten constant (Kм) was determined for all three substrates (glycine, GAB, ATP): glycine Kм increased by 30 - 115 fold, GAB Kм decreased by 8 - 17 fold, and the ATP Kм 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.
Contributing Partner: UNT College of Arts and Sciences