Reaction Mechanism of Glutathione Synthetase from Arabidopsis thaliana

Abstract

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...

Thiol-Based Regulation of Redox-Active Glutamate-Cysteine Ligase from Arabidopsis thaliana

“…Efforts to understand the regulation of glutathione biosynthesis in plants have primarily focused on the first three of these control mechanisms (Hell and Bergmann, 1990;May and Leaver, 1993;Foyer et al, 1997;May et al, 1998;Schafer et al, 1998;Xiang and Oliver, 1998;Xiang et al, 2001;Meyer and Fricker, 2002;Wachter et al, 2005;Herrera et al, 2007;Parisy et al, 2007). Interestingly, in Arabidopsis thaliana (thale cress) suspension cells, upregulation of GCL gene expression in response to various oxidative stresses was not observed, even though both GCL activity and cellular glutathione levels increased (May et al, 1998).…”

Structural Basis for Evolution of Product Diversity in Soybean Glutathione Biosynthesis

“…In the gEC binding site of hGS, Ser-176, Arg-295, Glu-241, and Gln-238 interact with the glutamyl portion of the molecule ( Figure 4A). Of these, the charge-charge interaction between the Arg and the carboxylate group is critical for gEC binding in GS (Herrera et al, 2007), suggesting an analogous role for this interaction in hGS. Similar to interactions observed in the structure of yeast GS complexed with gEC and an ATP analog (Gogos and Shapiro, 2002), Tyr-298 forms a hydrogen bond to the carbonyl of the glutamyl group and there is a bidentate charge-charge interaction between Arg-153 and the carboxylate of the cysteinyl moiety ( Figure 4A).…”

“…Steady state kinetic parameters of hGS for gEC, ATP, and b-Ala were determined (Table 1). In comparison to the GS from Arabidopsis Herrera et al, 2007), hGS displayed a turnover rate (V/E t ) fivefold lower but with comparable K m values for both ATP and gEC. In contrast with GS, which shows no activity if Gly is substituted with b-Ala, Ser, or Glu , hGS exhibited a 700-fold preference for b-Ala over Gly as the terminal substrate.…”

“…As noted for the bacterial and eukaryotic GS (Polekhina et al, 1999;Gogos and Shapiro, 2002;, nucleotide binding triggers movement of the lid domain and Ala-rich loop through multiple protein-ligand interactions. Based on the proposed reaction mechanism for GS (Herrera et al, 2007), enclosure of the active site likely prevents hydrolysis of the reactive acylphosphate intermediate ( Figure 1B). …”

“…The second step in the synthesis of either GSH or hGSH depends on the specificity of the synthetase for the terminal substrate. In nearly all organisms, glutathione synthetase (GS) catalyzes the addition of Gly to gEC (Meister, 1995;Herrera et al, 2007). In legumes, homoglutathione synthetase (hGS) uses b-Ala instead of Gly to form hGSH (Matamoros et al, 1999;Frendo et al, 2001;Iturbe-Ormaetxe et al, 2002).…”