Kinetic properties of missense mutations in patients with glutathione synthetase deficiency

Njålsson, Runa; Carlsson, Katarina Steen; Olin, B.; Carlsson, Birgit; Whitbread, Lel; Polekhina, Galina; Parker, M.W.; Norgren, Svante; Mannervik, Bengt; Board, Philip G.; Larsson, Agne

doi:10.1042/bj3490275

Cited by 14 publications

(9 citation statements)

References 12 publications

(25 reference statements)

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“…The steady-state kinetic parameters determined for AtGS (Table I) were similar to those reported for the GS from other eukaryotes (12,22,30). In contrast to earlier efforts (18 -19), degradation of AtGS was not observed during heterologous expression and subsequent purification of the protein (Fig.…”

Section: Resultsmentioning

confidence: 52%

“…However, the reaction rate was 2000-fold higher because of the greater purity of the AtGS sample. The k cat and K m values determined for each substrate of AtGS were equivalent to those reported for the yeast (12), human (22), and Plasmodium (30) enzymes. Compared with the E. coli GS (8), the K m values of AtGS for both ␥-glutamylcysteine and ATP were 10-fold lower, and the turnover rate was 10-fold slower.…”

Section: Resultsmentioning

confidence: 89%

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Kinetic Mechanism of Glutathione Synthetase from Arabidopsis thaliana

Jez

Cahoon

2004

Journal of Biological Chemistry

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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...

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Section: Resultsmentioning

confidence: 52%

Section: Resultsmentioning

confidence: 89%

Kinetic Mechanism of Glutathione Synthetase from Arabidopsis thaliana

Jez

Cahoon

2004

Journal of Biological Chemistry

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“…The protein is active as a homodimer with a subunit size of 77 kDa. The apparent K m value determined for ATP (59 µM) was found to be similar to the K app m value of the rat enzyme, whereas the K app m for glycine (5.0 mM) was four to five times higher when compared with mammalian GS [27,28]. The K app m for γ-GluAbu (107 µM) was six times lower when compared with the human enzyme but 2.5 times higher than the respective K m of the rat enzyme.…”

Section: Figure 3 Michaelis-menten and Lineweaver-burk Double-recipromentioning

confidence: 77%

“…PfGS was recombinantly expressed in E. coli BL 21 (DE3) and the purified protein had a specific activity of 5.24 units:mg ", which is comparable with that of the mammalian enzyme [27,28]. The protein is active as a homodimer with a subunit size of 77 kDa.…”

Section: Figure 3 Michaelis-menten and Lineweaver-burk Double-recipromentioning

confidence: 99%

Glutathione synthetase from Plasmodium falciparum

Meierjohann

Walter

Müller

2002

Biochemical Journal

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GSH is the major low-molecular-mass thiol in most organisms. The tripeptide maintains a reduced intracellular environment and protects cellular components from damaging oxidation. GSH is synthesized by the action of two ATP-dependent enzymic steps, in which γ-glutamylcysteine synthetase (γ-GCS) catalyses the ligation of glutamate and cysteine and subsequently glutathione synthetase (GS) adds glycine to the dipeptide. Recently it was shown that the synthesis of γ-glutamylcysteine is crucial for the survival of the erythrocytic stages of the malaria parasite Plasmodium falciparum by using the specific γ-GCS inhibitor buthionine sulphoximine. In order to investigate further the synthetic pathway of the tripeptide in the parasite, GS was cloned and expressed recombinantly. The deduced amino acid sequence of P. falciparum GS shares only a moderate degree of identity with other known GSs, but the residues responsible for substrate and co-factor binding are almost all conserved, with the exception of the ones involved in γ-glutamylcysteine binding. The protein is active as a dimer, with a subunit molecular mass of 77kDa, and the addition of reducing reagents such as dithiothreitol is essential in maintaining enzymic activity, indicating that thiol groups are important for stability and enzymic activity. The Kappm values for γ-glutamyl-α-aminobutyrate, ATP and glycine were determined to be 107.1μM, 59.1μM and 5.04mM, respectively, and the Vmax of 5.24±0.7μmol·min−1·mg−1 was in the same range as that of the mammalian enzymes. However, the negative co-operativity observed for γ-glutamylcysteine binding to the rat enzyme was not found for the parasite protein. This may be due to the alteration of several amino acids in the γ-glutamylcysteine-binding site.

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“…The cells were harvested in late log phase of growth by means of trypsin digestion, washed three times with phosphate buffered saline, and stored in liquid nitrogen until analyzed. Enzyme activity was measured as described (Njålsson et al, 2000). Protein content was determined using the Bradford method (Bio-Rad, Sundbyberg, Sweden).…”

Section: Fibroblast Culturing and Enzyme Activity In Crude Lysatesmentioning

confidence: 99%

Diagnostics in patients with glutathione synthetase deficiency but without mutations in the exons of the GSS gene

et al. 2003

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The synthesis of the ubiquitous tripeptide glutathione is impaired in patients with glutathione synthetase deficiency. The defect is inherited in an autosomal recessive manner, and the diagnosis is based on clinical, biochemical, and genetic criteria. In seven of our 30 index cases, however, no disease causing mutations could be identified in the coding exons or exon-intron boundaries of the glutathione synthetase gene GSS. These patients had severely decreased glutathione synthetase activities in lysates of cultured fibroblasts, and the levels of the enzyme were undetectable using a polyclonal antibody raised against human glutathione synthetase. RT-PCR mediated sequence analysis revealed previously not reported splice mutations in all patients. Thus, we conclude that in the investigation of patients with glutathione synthetase deficiency, and probably other genetic diseases as well, it might be time saving to initiate mutation analysis with sequencing of mRNA.

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Kinetic properties of missense mutations in patients with glutathione synthetase deficiency

Cited by 14 publications

References 12 publications

Kinetic Mechanism of Glutathione Synthetase from Arabidopsis thaliana

Kinetic Mechanism of Glutathione Synthetase from Arabidopsis thaliana

Glutathione synthetase from Plasmodium falciparum

Diagnostics in patients with glutathione synthetase deficiency but without mutations in the exons of the GSS gene

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