Glutathione is essential for maintaining the intracellular redox environment and is synthesized from ␥-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 ␥-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 ␥-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.Glutathione is a key modulator of the intracellular reducing environment that provides protection against reactive oxygen species and the detoxification of xenobiotics (1). In plants, metabolic pathways for the detoxification of herbicides, air pollutants such as sulfur dioxide, and heavy metals rely on glutathione (2-4). The biosynthesis of glutathione occurs through a twostep pathway found in mammals, bacteria, yeast, and plants. In the first reaction, glutamate-cysteine ligase (EC 6.3.2.2) catalyzes the ATP-dependent formation of the dipeptide ␥-glutamylcysteine from cysteine and glutamate (5). In the second step, glutathione synthetase (GS, 3 EC 6.3.2.3) catalyzes the addition of glycine to the dipeptide ( Fig. 1) (6). In this reaction, transfer of the ␥-phosphate of ATP to the C-terminal carboxylic acid of ␥-glutamylcysteine yields an acylphosphate intermediate. Nucleophilic attack on the acylphosphate intermediate by glycine results in formation of glutathione with release of ADP and inorganic phosphate (1, 7).The bacterial and eukaryotic GS form two distinct families that share no amino acid sequence homology. Structural and function...

Glutathione is found in mammals, plants, and bacteria and is synthesized by glutathione synthetase (GS). Some plants synthesize glutathione analogs in which ?‐alanine replaces glycine. The chemical diversity of these molecules suggests that the substrate specificity of the GS‐like peptide ligases differs from GS, but the molecular basis for this variety remains unclear. To better understand the reaction mechanism of Arabidopsis GS (AtGS) site‐directed mutagenesis was performed. Residues interacting with bound magnesium ions (E148, N150, E371, and E429) were essential for activity. K313 and K367 were important for ATP binding. Kinetic analysis suggests that R132 provides a key catalytic role. To define the determinants of substrate specificity, we compared AtGS (glycine specific) with Glycine max (soybean) homo‐glutathione synthetase (GmhGS; ?‐alanine specific). Sequence alignments suggest that two active site residues (AtGS: A466 and A467; GmhGS: L466 and P467) modulate substrate preference between glycine and ?‐alanine. The interconversion of AtGS to GmhGS and vice versa is described. GmhGS displays a 700‐fold preference for ?‐alanine, whereas, the double mutant accepts either substrate with equal efficiency. This suggests that differences outside the active site further define substrate specificity in this family of enzymes, and emphasize the need for crystallographic studies.