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, Michael W.; Norgren, Svante; Mannervik, Bengt; Board, Philip G.; Larsson, Agne

doi:10.1042/0264-6021:3490275

Cited by 19 publications

(16 citation statements)

References 13 publications

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“…Others, who extended their observations to lower, more physiological, substrate concentrations (< 0.4 mM) found non-linear plots indicative of allosteric activity, and estimated separate approximate K m values for the active sites of the enzyme (Luo et al, 2000;Njalsson et al, 2001;Oppenheimer et al, 1979). Our K m is higher than estimates for the initial K m for a comparable GS preparation and assay, but similar to K m estimates for the second binding site (K m2 = 1.50 mM; Dinescu et al, 2004), and to a reported value of 0.99+/-0.07 mM reported for GS purified from human blood haemolysates (Njålsson et al, 2000). K cat was higher than a value of 6.5 s -1 reported for a similar GS preparation analysed with 20 mM GAB as substrate at pH 8.2 (Dinescu et al, 2004), and closer to a more recently reported value of 19.5 s -1 (Slavens et al, 2011).…”

Section: Activity Of Gs Without Napqisupporting

confidence: 85%

The acetaminophen metabolite N-acetyl-p-benzoquinone imine (NAPQI) inhibits glutathione synthetasein vitro; a clue to the mechanism of 5-oxoprolinuric acidosis?

Walker

Mills²,

Anderson³

et al. 2016

Xenobiotica

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1. Metabolic acidosis due to accumulation of l-5-oxoproline is a rare, poorly understood, disorder associated with acetaminophen treatment in malnourished patients with chronic morbidity. l-5-Oxoprolinuria signals abnormal functioning of the γ-glutamyl cycle, which recycles and synthesises glutathione. Inhibition of glutathione synthetase (GS) by N-acetyl-p-benzoquinone imine (NAPQI) could contribute to 5-oxoprolinuric acidosis in such patients. We investigated the interaction of NAPQI with GS in vitro. 2. Peptide mapping of co-incubated NAPQI and GS using mass spectrometry demonstrated binding of NAPQI with cysteine-422 of GS, which is known to be essential for GS activity. Computational docking shows that NAPQI is properly positioned for covalent bonding with cysteine-422 via Michael addition and hence supports adduct formation. 3. Co-incubation of 0.77 μM of GS with NAPQI (25-400 μM) decreased enzyme activity by 16-89%. Inhibition correlated strongly with the concentration of NAPQI and was irreversible. 4. NAPQI binds covalently to GS causing irreversible enzyme inhibition in vitro. This is an important novel biochemical observation. It is the first indication that NAPQI may inhibit glutathione synthesis, which is pivotal in NAPQI detoxification. Further studies are required to investigate its biological significance and its role in 5-oxoprolinuric acidosis.

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Section: Activity Of Gs Without Napqisupporting

confidence: 85%

The acetaminophen metabolite N-acetyl-p-benzoquinone imine (NAPQI) inhibits glutathione synthetasein vitro; a clue to the mechanism of 5-oxoprolinuric acidosis?

Walker

Mills²,

Anderson³

et al. 2016

Xenobiotica

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“…Since negative cooperativity supposedly makes enzymes less sensitive to changes in substrate concentrations, the allostery would counteract such fluctuations and emphasizes the importance of keeping GSH production constant. Kinetic analyses of recombinant human GSS containing naturally occurring missense mutations showed that these affect the stability, catalytic capacity, substrate affinity and cooperativity of the enzyme [78,79].…”

Section: Kinetics Of Gssmentioning

confidence: 99%

Glutathione synthetase deficiency

Njålsson

2005

Cell. Mol. Life Sci.

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Glutathione (GSH), one of the most important antioxidants in the eukaryotic organism, is synthesized in a two-step procedure where the last step is catalysed by the enzyme glutathione synthetase (GSS). GSS deficiency is inherited autosomal recessively, and patients with this disease can be divided into three groups, according to their clinical phenotype. Mildly affected patients have mutations affecting the stability of the enzyme, causing a compensated haemolytic anaemia; moderately affected patients have, in addition, metabolic acidosis; and severely affected patients also develop neurological defects and show increased susceptibility to bacterial infections. Moderately and severely affected patients have mutations that compromise the catalytic properties of the enzyme. 5-Oxoprolinuria appears in all three groups, but is more pronounced in the two latter groups. Today, no cure can be offered these patients; they are given vitamins C and E to boost their antioxidant levels, and bicarbonate to correct metabolic acidosis.

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“…GS activity GS activity in cultured fibroblasts was analysed as described (Nja˚lsson et al 2000) or obtained from previous reports (Shi et al 1996;Dahl et al 1997;Ristoff et al 2001Nja˚lsson et al 2003). In six of the patients, no GS activity data in cultured fibroblasts were available.…”

Section: Genotyping and Haplotypingmentioning

confidence: 99%

Genotype, enzyme activity, glutathione level, and clinical phenotype in patients with glutathione synthetase deficiency

et al. 2005

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Glutathione synthetase (GS) deficiency is a rare autosomal recessive disorder. The clinical phenotype varies widely, and nearly 30 different mutations in the GSS gene have been identified. In the present study, genotype, enzyme activity, metabolite levels and clinical phenotype were evaluated in 41 patients from 33 families. From some of the patients, data on glutathione (GSH) levels and gamma-glutamylcysteine levels in cultured fibroblasts were also available. Twenty-seven different mutations were found: 14 missense, 9 splice, 2 deletions, 1 insertion and 1 nonsense mutation. Twenty-three patients were homozygous and 18 were compound heterozygous. The moderate and severe clinical phenotypes could not be distinguished based on enzyme activity, GSH or gamma-glutamylcysteine levels in cultured fibroblasts. However, in fibroblasts, the residual GS activity was correlated with the GSH level. All mutations causing frameshifts, premature stop codons or aberrant splicing were associated with moderate or severe clinical phenotypes including haemolytic anaemia, 5-oxoprolinuria, and (in several forms) neurodevelopmental signs. The data indicate that additional genetic or environmental factors modify at least the moderate and severe phenotypes and that the clinical classification given to the patients may be influenced by variation in follow-up. The type of mutation involved can, to some extent, predict a mild versus a more severe phenotype.

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Kinetic properties of missense mutations in patients with glutathione synthetase deficiency

Cited by 19 publications

References 13 publications

The acetaminophen metabolite N-acetyl-p-benzoquinone imine (NAPQI) inhibits glutathione synthetasein vitro; a clue to the mechanism of 5-oxoprolinuric acidosis?

The acetaminophen metabolite N-acetyl-p-benzoquinone imine (NAPQI) inhibits glutathione synthetasein vitro; a clue to the mechanism of 5-oxoprolinuric acidosis?

Glutathione synthetase deficiency

Genotype, enzyme activity, glutathione level, and clinical phenotype in patients with glutathione synthetase deficiency

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