The Journal of biological chemistry

Reaction mechanism of glutathione synthetase from Arabidopsis thaliana: site-directed mutagenesis of active site residues.

PMID 17452339


Glutathione is essential for maintaining the intracellular redox environment and is synthesized from gamma-glutamylcysteine, glycine, and ATP by glutathione synthetase (GS). To examine the reaction mechanism of a eukaryotic GS, 24 Arabidopsis thaliana GS (AtGS) mutants were kinetically characterized. Within the gamma-glutamylcysteine/glutathione-binding site, the S153A and S155A mutants displayed less than 4-fold changes in kinetic parameters with mutations of Glu-220 (E220A/E220Q), Gln-226 (Q226A/Q226N), and Arg-274 (R274A/R274K) at the distal end of the binding site resulting in 24-180-fold increases in the K(m) values for gamma-glutamylcysteine. Substitution of multiple residues interacting with ATP (K313M, K367M, and E429A/E429Q) or coordinating magnesium ions to ATP (E148A/E148Q, N150A/N150D, and E371A) yielded inactive protein because of compromised nucleotide binding, as determined by fluorescence titration. Other mutations in the ATP-binding site (E371Q, N376A, and K456M) resulted in greater than 30-fold decreases in affinity for ATP and up to 80-fold reductions in turnover rate. Mutation of Arg-132 and Arg-454, which are positioned at the interface of the two substrate-binding sites, affected the enzymatic activity differently. The R132A mutant was inactive, and the R132K mutant decreased k(cat) by 200-fold; however, both mutants bound ATP with K(d) values similar to wild-type enzyme. Minimal changes in kinetic parameters were observed with the R454K mutant, but the R454A mutant displayed a 160-fold decrease in k(cat). In addition, the R132K, R454A, and R454K mutations elevated the K(m) value for glycine up to 11-fold. Comparison of the pH profiles and the solvent deuterium isotope effects of A. thaliana GS and the Arg-132 and Arg-454 mutants also suggest distinct mechanistic roles for these residues. Based on these results, a catalytic mechanism for the eukaryotic GS is proposed.

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γ-Glu-Cys, ≥80% (HPLC)