Kinetic Mechanism of Glutathione Synthetase from Arabidopsis thaliana

Abstract

Glutathione synthetase (GS) catalyzes the ATP-dependent formation of the ubiquitous peptide glutathione from ␥-glutamylcysteine and glycine. The bacterial and eukaryotic GS form two distinct families lacking amino acid sequence homology. Moreover, the detailed kinetic mechanism of the bacterial and the eukaryotic GS remains unclear. Here we have overexpressed Arabidopsis thaliana GS (AtGS) in an Escherichia coli expression system and purified the recombinant enzyme for biochemical characterization. AtGS is functional as a homodimeric protein with steady-state kinetic properties similar to those of other eukaryotic GS. The kinetic mechanism of AtGS was investigated using initial velocity methods and product inhibition studies. The best fit of the observed data was to the equation for a random Ter-reactant mechanism in which dependencies between the binding of some substrate pairs were preferred. The binding of either ATP or ␥-glutamylcysteine increased the binding affinity of AtGS for the other substrate by 10-fold. Likewise, the binding of ATP or glycine increased binding affinity for the other ligand by 3.5-fold. In contrast, binding of either glycine or ␥-glutamylcysteine causes a 6.7-fold decrease in binding affinity for the second molecule. Product inhibition studies suggest that ADP is the last product released from the enzyme. Overall, these observations are consistent with a random Ter-reactant mechanism for the eukaryotic GS in which the binding order of certain substrates is kinetically preferred for catalysis.Glutathione is a key modulator of the intracellular reducing environment that provides protection against reactive oxygen species and maintains protein thiols in a reduced state (1). In plants, metabolic pathways for the detoxification of herbicides (2), air pollutants such as sulfur dioxide and ozone (3), and heavy metals (4) also rely on glutathione. For example, glutathione is the metabolic precursor for the synthesis of phytochelatin peptides that sequester cadmium, mercury, and arsenic (5).Glutathione biosynthesis occurs through a two-step pathway found in all organisms. 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. Next, glutathione synthetase (GS, 1 EC 6.3.2.3) catalyzes the addition of glycine to the dipeptide (Scheme I).In this reaction, transfer of the ␥-phosphate group of ATP to the C-terminal carboxylate of ␥-glutamylcysteine yields an acylphosphate intermediate. Nucleophilic attack on the acylphosphate intermediate by glycine leads to formation of glutathione with release of ADP and inorganic phosphate (1, 6). GS from bacteria and eukaryotes form two distinct families that share no amino acid sequence homology (7). Detailed characterization of the Escherichia coli GS demonstrates that this enzyme is functional as a tetramer of ϳ300-residue subunits (8 -11), whereas the mammalian and yeast GS are active as dimers of ϳ470-residue subunits (12-15). Despite the lack of se...

Two Chimeric Regulators of G-protein Signaling (RGS) Proteins Differentially Modulate Soybean Heterotrimeric G-protein Cycle

“…The RGS domains of GmRGS1 and GmRGS2 (amino acids 251-464) were cloned into pET-28a (Novagen, WI) and transformed into E. coli Rosetta cells (Novagen). Recombinant proteins were purified using Ni 2ϩ -affinity chromatography (32). Protein aliquots were snap-frozen in liquid nitrogen and stored at Ϫ80°C.…”

Reaction Mechanism of Glutathione Synthetase from Arabidopsis thaliana

“…Enzyme Assays-Enzymatic activity of GS was determined spectrophotometrically by measuring the rate of ADP formation using a coupled assay with pyruvate kinase and lactate dehydrogenase (6). Standard reactions were performed at 25°C in a 0.5-ml reaction volume containing 100 mM HEPES (pH 7.5), 150 mM NaCl, 20 mM MgCl 2 , 2.5 mM ␥-glutamylcysteine, 10 mM glycine, 2.5 mM disodium ATP, 2 mM sodium phosphoenolpyruvate, 0.2 mM NADH, 5 units of type III rabbit muscle pyruvate kinase, and 10 units of type II rabbit muscle lactate dehydrogenase.…”

“…Mutagenesis, Expression, and Purification-Site-directed mutants of AtGS (R132A, R132K, E148A, E148Q, N150D, N150A, S153A, S155A, E220Q, E220A, Q226N, Q226A, R274K, R274A, K313M, K367M, N376A, E371Q, E371A, E429Q, E429A, R454A, R454K, and K456M) were generated using oligonucleotides containing the desired mutation and the QuikChange PCR method (Stratagene) with the pET28a-AtGS expression vector as template (6). Automated nucleotide sequencing confirmed the fidelity of the mutant gene sequences (Washington University Sequencing Facility, St. Louis).…”

“…Automated nucleotide sequencing confirmed the fidelity of the mutant gene sequences (Washington University Sequencing Facility, St. Louis). Recombinant proteins were overexpressed in E. coli C41(DE3) and were purified by Ni 2ϩ affinity and size-exclusion chromatographies, as described previously (6).…”

“…In contrast, the GS from mammals, yeast, and plants, which are related by ϳ30% amino acid sequence identity ( Fig. 2A), are active as homodimeric enzymes, with each monomer consisting of ϳ470 amino acids (6,(13)(14)(15)(16). Despite the lack of sequence homology and different oligomeric structures, the E. coli, human, and yeast GS share a common structural fold and are part of the ATP-grasp structural family (17,18).…”