Tripeptide (Glutathione) Synthetase. Purification, Properties, and Mechanism of Action<sup>*</sup>

Mooz, Elizabeth Dodd; Meister, Alton

doi:10.1021/bi00858a022

Cited by 81 publications

(26 citation statements)

References 20 publications

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“…Glutathione synthetase activity was determined essentially as described previously (15)(16)(17). The reaction mixtures (final volume, 0.25 ml) contained the cell extract, i-'y-glutamyl->La-aminobutyrate,** (1.5 umoles), [1-'4C]glycine (4 Imoles; 55,000 cpm/lumole), sodium ATP (1 smole), sodium phosphoenolpyruvate (1 smole), pyruvate kinase (4 ,.g), Tris-HCl buffer (25 ,moles; pH 8.2), potassium chloride (25 ,moles), magnesium chloride (2 jsmoles), and bovineserum albumin (0.25 mg).…”

Section: Methodsmentioning

confidence: 99%

Glutathione Synthetase Deficiency, an Inborn Error of Metabolism Involving the γ-Glutamyl Cycle in Patients with 5-Oxoprolinuria (Pyroglutamic Aciduria)

Wellner

Sekura

Meister

et al. 1974

Proc. Natl. Acad. Sci. U.S.A.

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135

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Enzyme studies on placenta, cultured skin fibroblasts, and erythrocytes from two sisters with the inborn error 5-oxoprolinuria (pyroglutamic aciduria) indicate that the metabolic lesion in this disease is at the glutathione syntbetase (EC 6.3.2.3) step of the 7y-glutamyl cycle. Excessive urinary excretion of 5-oxoproline by these patients appears to be associated with increased synthesis of -y-glutamyl-cysteine and formation of 5-oxoproline from this dipeptide. Thus, 5-oxoproline is produced in amounts that exceed the normal capacity of 5-oxoprolinase to convert it to glutamate. The data indicate that it may be possible to identify individuals who are heterozygous for this trait by determinations of erythrocyte glutathione synthetase.Previous studies on glutathione have indicated that its synthesis and utilization are integrated in a series of enzymecatalyzed reactions, which have been termed the y-glutamyl cycle. The y-glutamyl cycle consists of six enzyme-catalyzed reactions ( Fig. 1): amino acid-dependent breakdown of glutathione to yield y-glutamyl amino acid and cysteinyl-glycine catalyzed by -y-glutamyl transpeptidase (EC 2.3.2.2) conversion of -y-glutamyl amino acid to 5-oxoproline4 and the corresponding free amino acid catalyzed by 'y-glutamyl cyclotransferase (EC 2.3.2.4), enzymatic hydrolysis of cysteinylglycine to cysteine and glycine, conversion of 5-oxoproline to glutamate catalyzed by 5-oxoprolinase, and the two-step synthesis of glutathione from glutamate, cysteine, and glycine catalyzed by -y-glutamyl-cysteine synthetase (EC 6.3.2.2) and glutathione (GSH) synthetase (EC 6.3.2.3). The evidence for the existence of the cycle and for its involvement in amino-acid transport has been reviewed recently (1-3). §In 1970 Jellum et al. (5) reported on an interesting patient who had a massive urinary excretion of 5-oxoproline (about 30 g/day compared to less than a few mg/day in normal individuals).f This patient was a 19-year-old male with metabolic acidosis and multiple neurological symptoms such as mental retardation, spastic tetraparesis, and ataxia. The patient was at first thought to have a defect in the urea cycle (5), but subsequent studies (7-10) have indicated a lesion in the y-glutamyl cycle, although the exact location was not determined.A (12). Since 1 year of age she is known to have excreted in the urine more than 6 g of 5-oxoproline per day, and her plasma concentration of 5-oxoproline has been 2-5 mM (11,12). Studies in which this patient was given [14C]5-oxoproline are consistent with the conclusion that about 25% of the amount of 5-oxoproline formed by this patient is excreted and that the rernainder is metabolized (11); a similar conclusion may now be drawn from the studies done on the first patient (8).Studies on the peripheral leukocytes of the second patient have indicated the presence of 5-oxoprolinase (11). The young girl with 5-oxoprolinuria has recently acquired a sister who suffers from the same inborn error of metabclism (12). 13oth patients, in addition to increased co...

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Section: Methodsmentioning

confidence: 99%

Glutathione Synthetase Deficiency, an Inborn Error of Metabolism Involving the γ-Glutamyl Cycle in Patients with 5-Oxoprolinuria (Pyroglutamic Aciduria)

Wellner

Sekura

Meister

et al. 1974

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

135

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“…There is some similarity between the amino acid compositions of the two enzymes with essentially identical contents of isoleucine, histidine, proline, and glutamic acid. All of the remaining amino acids are present in both enzymes in amounts which agree within 20% with the exception of i cystine, methionine, lysine, and arginine (4). The Michaelis constants for glycine and GC were essentially identical comparing yeast GSH synthetase with the erythocyte enzyme, while the Km for ATP was 3-fold higher with the latter enzyme (4).…”

Section: Methodsmentioning

confidence: 73%

“…The Michaelis constants for glutamate and for ATP are quite similar for enzyme from wheat germ and human erythrocytes while the Km for cysteine was 10-fold higher (4 X 10' moles/liter) in wheat germ than in erythrocytes. GSH synthetase has been previously purified from yeast (2,4) and pigeon liver (1). The homogeneous yeast enzyme is striking in that the intrinsic specific activity is 20-fold higher than that of the erythrocyte enzyme.…”

Section: Methodsmentioning

confidence: 99%

Glutathione Synthesis in Human Erythrocytes

Majerus¹,

Brauner²,

Smith³

et al. 1971

J. Clin. Invest.

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“…Cell disruption was performed in a cell mill using glass beads (3 ϫ 3 min dry; 6 ϫ 3 min with 10 ml of immobilized metal ion affinity chromatography (IMAC) binding buffer/g of lyophilized cells). After centrifugation (30 min, 1500 ϫ g, SS34 rotor, 4°C) the supernatant was utilized for the following purification GSH2-Fwd 5Ј-AAA GGA TCC ATG GAA ATT GAG AAG TAT ACA CCG GAG-3Ј BamHI GSH2-Rev 5Ј-AAA CCC GGG TTA ATG ATG ATG ATG ATG ATG TTC AGA AAG TTC AAT ACT AG-3Ј SmaI GSH2M214F 5Ј-AAA GGA TCC ATG AAT ATT GCT TCT GAT AAC-3Ј BamHI GSH2K213R 5Ј-AAA CCC GGG TTA TTT GCT TGT AAT GTT TTT AAC GTA ATC ACG-3Ј SmaI CWGLU44A 5Ј-AAA GAA TTC TCA AAT ACA TGT ATA ATT TT-3Ј EcoRI GSH2Irev 5Ј-AAA GGT ACC GTA GGC ATC TAC AGC ATT-3Ј KpnI GSH2IIFwd 5Ј-AAA GGT ACC GAT AAC ACG AAA CCC ATT-3Ј KpnI CW8DDA 5Ј-AAA GGA TCC AAT ATC TTC TTT GAG GCA C-3Ј BamHI PermutGr 5Ј-AAA GGT ACC TTC AGA AAG TTC AAT ACT AG- 4 , 500 mM NaCl, 10 mM imidazole, pH 7.4), the bound proteins were eluted by increasing the imidazole concentration stepwise to 50 mM and then to 100 mM. Glutathione synthetase eluted at an imidazole concentration of 100 mM.…”

Section: Methodsmentioning

confidence: 99%

The Glutathione Synthetase of Schizosaccharomyces pombe Is Synthesized as a Homodimer but Retains Full Activity When Present as a Heterotetramer

Phlippen¹,

Hoffmann²,

Fischer³

et al. 2003

Journal of Biological Chemistry

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Glutathione synthetase was overexpressed as a histidine-tagged protein in Schizosaccharomyces pombe and purified by two-step affinity chromatography. The recovered enzyme occurred in two different forms: a homodimeric protein consisting of two identical 56-kDa subunits and a heterotetrameric protein composed of two 32-kDa and two 24-kDa subfragments. Both forms are encoded by the GSH2 gene. The 56-Da protein corresponds to the complete GSH2 open reading frame, while the subfragments are produced following the cleavage of this larger protein by a metalloprotease. A stable homodimer was obtained by site-directed mutagenesis to remove the protease cleavage site, and this showed normal activity. A structural model of the fission yeast glutathione synthetase was produced, based on the x-ray coordinates of the human enzyme. According to this model the interacting domains of the proteolytic subfragments are strongly entangled. The subfragments were therefore coexpressed as independent proteins. These subfragments assembled correctly to yield functional heterotetramers with equivalent activity to the wild type enzyme. Furthermore, a permuted version of the protein was created. This also showed normal levels of glutathione synthetase activity. These data provide novel insight into the mechanisms of protein folding and the structure and evolution of the glutathione synthetase family. Glutathione (GSH,1 ␥-Glu-Cys-Gly) is a biologically important thiol found in almost all procaryotic and eucaryotic cells. It has been assigned several cellular functions, including protection against oxidative damage, maintenance of a reducing cellular thiol-disulfide balance, electron donation for a number of enzymes, protection of protein sulfhydryls from irreversible oxidation, and detoxification of foreign compounds (1-3). Glutathione is synthesized enzymatically from its constituent amino acids in two consecutive reactions. Glutathione synthetase (EC 6.3.2.3) catalyzes the second step, the addition of glycine to ␥-glutamylcysteine.In most eucaryotic organisms, including Saccharomyces cerevisiae, Arabidopsis thaliana, Homo sapiens, and Rattus norvegicus, glutathione synthetase is a 104 -112-kDa homodimer composed of two identical 52-56-kDa subunits (4 -10). In contrast, the structure of the Schizosaccharomyces pombe enzyme has not been completely elucidated. The enzyme was purified as a 120-kDa heterotetramer consisting of two kinds of subunits, called the "small" and "large" subunits, with apparent molecular masses of 26 and 33 kDa (11). While the gene encoding the 26-kDa subunit was unknown, the gene encoding the 33-kDa subunit, GSH2, had been cloned, and the coding region had been assigned to the 3Ј end (12). When the GSH2 gene was re-sequenced at a later date, however, frameshift errors in the original sequence were identified (13,14). These errors resulted in a shortened open reading frame, downstream from the correct start codon. A protein derived from the full-length GSH2 gene would have a molecular mass of 56 kDa, and BLAST search...

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Tripeptide (Glutathione) Synthetase. Purification, Properties, and Mechanism of Action^*

Cited by 81 publications

References 20 publications

Glutathione Synthetase Deficiency, an Inborn Error of Metabolism Involving the γ-Glutamyl Cycle in Patients with 5-Oxoprolinuria (Pyroglutamic Aciduria)

Glutathione Synthetase Deficiency, an Inborn Error of Metabolism Involving the γ-Glutamyl Cycle in Patients with 5-Oxoprolinuria (Pyroglutamic Aciduria)

Glutathione Synthesis in Human Erythrocytes

The Glutathione Synthetase of Schizosaccharomyces pombe Is Synthesized as a Homodimer but Retains Full Activity When Present as a Heterotetramer

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Tripeptide (Glutathione) Synthetase. Purification, Properties, and Mechanism of Action*

Cited by 81 publications

References 20 publications

Glutathione Synthetase Deficiency, an Inborn Error of Metabolism Involving the γ-Glutamyl Cycle in Patients with 5-Oxoprolinuria (Pyroglutamic Aciduria)

Glutathione Synthetase Deficiency, an Inborn Error of Metabolism Involving the γ-Glutamyl Cycle in Patients with 5-Oxoprolinuria (Pyroglutamic Aciduria)

Glutathione Synthesis in Human Erythrocytes

The Glutathione Synthetase of Schizosaccharomyces pombe Is Synthesized as a Homodimer but Retains Full Activity When Present as a Heterotetramer

Contact Info

Product

Resources

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Tripeptide (Glutathione) Synthetase. Purification, Properties, and Mechanism of Action^*