Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.3.2.3 (glutathione synthetase)
678 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glutathione (GSH) synthetase [gamma-L-glutamyl-L-cysteine:glycine ligase (ADP-forming), EC 6.3.2.3], an enzyme present in almost all cells, catalyzes the ATP-dependent synthesis of GSH from gamma-L-glutamyl-L-cysteine and glycine. Highly purified preparations of the enzyme have been obtained from rat kidney and several lower forms. The rat kidney enzyme (M(r), 118,000), which contains approximately 2% carbohydrate, is composed of two apparently identical subunits. The cDNA encoding rat kidney GSH synthetase was isolated from a rat kidney lambda gt11 cDNA library by immunoscreening with an antibody prepared against the isolated enzyme. The cDNA contains 1905 nucleotides and an open reading frame of 1422 nucleotides coding for 474 amino acids. The cDNA has a 3' untranslated region of 439 nucleotides, which includes a poly(A) tail. The deduced amino acid sequence (M(r), 52,344) contains all five of the peptide sequences that were independently determined by Edman degradation. The cDNA was expressed in Escherichia coli. The amino acid sequence of the rat kidney enzyme has no significant similarity to that of the enzyme from E. coli and shows some similarity to those deduced for the yeast and frog enzymes. Knowledge of this amino acid sequence is expected to facilitate elucidation of the sequence of the corresponding human enzyme and to lead to studies on the biochemical mechanisms involved in human GSH synthetase deficiency as well as to development of improved methods for prenatal diagnosis of these inborn diseases.
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PMID:Amino acid sequence of rat kidney glutathione synthetase. 786 66

Parenteral administration of ferric nitrilotriacetate (Fe-NTA), a known carcinogen in mouse and rat kidneys, enhances iron-dependent lipid peroxidation (LP) and causes acute renal tubular necrosis. We assume that filtered Fe-NTA in vivo is rapidly reduced by cysteine, a component of glutathione which is hydrolyzed by gamma-GTP and dipeptidase, and that this reduced iron initiates lipid peroxidation in the lumen. In addition, the fatty acid composition of phospholipids between the cortex and the medulla may differ, because only the proximal tubules (which are located mainly in the cortex) are known to be vulnerable to LP. We tested these assumptions in the present study. Gas chromatographic determination of fatty acid composition in five male and five female 6-week-old normal ddY mice showed the ratio of polyunsaturated fatty acids to saturated fatty acids plus C18: 1, a single double-bond fatty acid, to be 0.98 +/- 0.08 (av +/- SD) in the male cortex and 1.00 +/- 0.08 in the female cortex. In the male and female medulla, however, it was 0.78 +/- 0.09 (P < 0.05, vs cortex) and 0.68 +/- 0.04 (P < 0.01, vs cortex), respectively. Pretreatment of the animals with buthionine sulfoximine, a glutathione synthetase inhibitor, and a procedure that reduces total glutathione content in the kidneys, suppressed LP. Reduced thiobarbituric acid reactive substances were also observed in animals treated with AT-125, a gamma-GTP inhibitor, and in animals with immature gamma-GTP activity. These results are consistent with our assumptions.
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PMID:Glutathione cycle dependency of ferric nitrilotriacetate-induced lipid peroxidation in mouse proximal renal tubules. 809 33

The catalytic mechanism of glutathione synthetase is proposed to proceed via phosphorylation of the dipeptide substrate to yield an acyl phosphate intermediate; this intermediate is subsequently attacked by glycine, followed by loss of inorganic phosphate, leading to glutathione formation. A flexible loop (Ile226-Gly242) in Escherichia coli B glutathione synthetase is proposed to stabilize the acyl phosphate intermediate by preventing its decomposition by hydrolysis with water [Tanaka, T., Kato, H., Nishioka, T., & Oda, J. (1992) Biochemistry 31, 2259-2265; Tanaka, T., Yamaguchi, H., Kato, H., Nishioka, T., Katsube, Y., & Oda, J. (1993) Biochemistry 32, 12398-12404]. To investigate the function of the loop in the E. coli enzyme definitely, a loopless mutant in which the loop (Ile226-Arg241) was replaced with three residues of glycine was constructed. The crystal structure of the loopless mutant enzyme was essentially identical with that of the wild-type enzyme. Kinetic measurements showed that the replacement of the loop led to increases in the Km values, especially for the glycine, and a 930-fold decrease in the k0 value. Hence, the loopless mutant was 3 x 10(4) less active in terms of its specificity constant (k0/Km) for glycine than the wild-type enzyme. Moreover, the loopless mutant showed gamma-L-glutamyl-L-cysteine-dependent ATP hydrolase activity to almost the same extent as its glutathione synthetase activity. These studies support the fact that the loop enhances the recognition of glycine as well as stabilizes the acyl phosphate intermediate so that the intermediate rapidly reacts with glycine.
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PMID:Flexible loop that is novel catalytic machinery in a ligase. Atomic structure and function of the loopless glutathione synthetase. 817 74

A fluorimetric technique previously described for other tissues has been applied to determine levels of glutathione and its synthetic rates in ocular tissues of Hartley guinea-pigs. Monochlorobimane forms a stable, fluorescent adduct with glutathione in a reaction catalyzed by glutathione-S-transferase. The fluorescent signal recorded over time is directly proportional to the synthetic rate of glutathione. Lens, cornea and retina were homogenized and cytosolic fractions dialyzed overnight to deplete endogeneous glutathione. Glutathione synthetic rates were determined from a mixture of glutathione precursors and co-factors, viz. cysteine+dithiothreitol, glutamate+glycine, ATP and Mg++ in the presence of monochlorobimane. The mixture was supplemented with glutathione-S-transferase to catalyze the formation of the fluorescent adduct. Glutathione synthetic rates were determined in the absence and presence of buthionine sulfoximine, an inhibitor of gamma-glutamyl cysteine synthetase. The difference in fluorescence change over time in the presence and absence of buthionine sulfoximine was used to estimate glutathione synthesis. Basal levels of glutathione in pre-dialyzed cytosolic fractions of the lens, cornea, and retina were 21.8 +/- 2.2, 36.5 +/- 4.1 and 38.6 +/- 2.8 nmol mg-1 protein, respectively. The maximal glutathione synthetic rates in these tissues were 0.52 +/- 0.04, 2.25 +/- 0.67 and 3.35 +/- 0.65 nmol min-1 mg-1 protein, respectively. When gamma-glutamyl cysteine is used as a precursor instead of cysteine, the glutathione synthetase activities from lenses and retinas were 0.19 +/- 0.08 and 1.54 +/- 0.76 nmol-1 min mg-1 protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A simple technique to determine glutathione (GSH) levels and synthesis in ocular tissues as GSH-bimane adduct: application to normal and galactosemic guinea-pigs. 843 34

Glutathione synthetase (gamma-L-glutamyl-L-cysteine: glycine ligase (ADP-forming) EC 6.3.2.3: GSHase) catalyzes the synthesis of glutathione from gamma-L-glutamyl-L-cysteine and Gly in the presence of ATP. The enzyme from Escherichia coli is a tetramer with four identical subunits of 316 amino acid residues. The crystal structure of the enzyme has been determined by isomorphous replacement and refined to a 2.0 A resolution. Two regions, Gly164 to Gly167 and Ile226 to Arg241, are invisible on the electron density map. The refined model of the subunit includes 296 amino acid residues and 107 solvent molecules. The crystallographic R-factor is 18.6% for 17.914 reflections with F > 3 sigma between 6.0 A and 2.0 A. The structure consists of three domains: the N-terminal, central, and C-terminal domains. In the tetrameric molecule, two subunits that are in close contact form a tight dimer, two tight dimers forming a tetramer with two solvent regions. The ATP molecule is located in the cleft between the central and C-terminal domains. The ATP binding site is surrounded by two sets of the structural motif that belong to those respective domains. Each motif consists of an anti-parallel beta-sheet and a glycine-rich loop.
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PMID:Three-dimensional structure of the glutathione synthetase from Escherichia coli B at 2.0 A resolution. 844 37

Monocrotaline (MONO), a pyrrolizidine alkaloid, causes veno-occlusive disease of the liver, pulmonary arterial hypertension, and right ventricular hypertrophy. Toxicity is due to the hepatic formation of a pyrolic metabolite that can be detoxified by conjugation with glutathione (GSH). We have shown that the GSH content of the liver affects the quantity of the pyrrolic metabolite that is released from the liver. We have now examined whether MONO, in turn, affects GSH metabolism. Twenty-four hours after administration of MONO to rats (65 mg/kg, i.p.), the highest concentration of bound pyrrolic metabolites was found in the liver, followed by the lung and kidney. Heart and brain contained lower concentrations of these metabolites. Significantly higher levels of GSH were found in liver and lungs of MONO-treated rats than in saline-injected control animals. In the liver, activities of the following enzymes were elevated: gamma-glutamylcysteine synthetase, GSH synthetase, gamma-glutamyl transpeptidase, dipeptidase, and microsomal GSH transferase. The same changes were seen in the lung. In the heart, gamma-glutamyl transpeptidase activity was decreased markedly, and cytosolic GSH transferase activity was elevated. In the kidney, the activities of GSH synthetase, gamma-glutamyl transpeptidase, and cytosolic GSH transferase were increased. Our results establish a mutual interaction of MONO and sulfur metabolism. It appears that an early metabolic action of MONO is to modify sulfur amino acid metabolism, diverting cysteine metabolism from oxidation to taurine towards synthesis of GSH.
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PMID:Effects of monocrotaline, a pyrrolizidine alkaloid, on glutathione metabolism in the rat. 857 5

Lys18, Arg86, Asn283, Ser286, Thr288 and Glu292 of glutathione synthetase from Escherichia coli B are presumed to be highly concerned with the substrate, gamma-L-glutamyl-L-cysteine (gamma-Glu-Cys), binding by X-ray crystallography and affinity labeling studies. Using site-directed mutagenesis, we investigated functional roles of those residues for gamma-Glu-Cys binding. The mutant enzymes of Arg86 and Asn283 altered their kinetic parameters, especially the Michaelis constants of gamma-Glu-Cys. In the case of Asn283, the residue is not likely to have an essential role in gamma-Glu-Cys binding but its side chain would extend to make a van der Waals contact with bound gamma-Glu-Cys. Chemical modification of a cysteine residue with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) showed Arg86 would not only be much responsible for gamma-Glu-Cys binding but would also have a role in maintaining the structural integrity of the enzyme. The other mutant enzymes showed little defect in their kinetic parameters of gamma-Glu-Cys.
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PMID:Site-directed mutagenesis of glutathione synthetase from Escherichia coli B: mapping of the gamma-L-glutamyl-L-cysteine-binding site. 857 99

Eisai hyperbilirubinemic rats (EHBR) are mutant Sprague-Dawley rats that exhibit impaired biliary organic anion and reduced glutathione (GSH) secretion. In addition, liver GSH levels are twice that of age-matched controls. The mechanisms for the defect in biliary GSH secretion and the increase in cell GSH are not fully understood. We previously showed that canalicular membrane-enriched vesicles isolated from EHBR livers exhibited normal GSH transport. In the present study, we examined the steady-state rat canalicular reduced glutathione transporter (RcGshT) messenger RNA (mRNA) and protein levels, as well as the mechanisms for the increase in cell GSH. Both Northern and Western blot analyses of EHBR livers showed nearly identical RcGshT mRNA and polypeptide levels, respectively, as compared with controls. Treatment with phenobarbital, which increased steady-state RcGshT mRNA by five- to sixfold, RcGshT polypeptide, and biliary GSH secretion by onefold in controls, had a smaller effect on steady-state RcGshT-mRNA level in EHBR (by 1.5-fold) and did not increase RcGshT polypeptide or biliary GSH secretion. In examining possible mechanisms for increased liver GSH, both cysteine level and gamma-glutamylcysteine synthetase (GCS) activity were significantly higher than controls, while the activity of GSH synthetase was unchanged. Northern and Western blot analyses also showed increased steady-state GCS heavy subunit (GCS-HS) mRNA and polypeptide levels, respectively. In addition to liver, GSH levels in kidney, duodenal, jejunal, and ileal mucosa of EHBR were 200% to 300% of age-matched control rats. GCS activity was also increased in kidney cytosol of EHBR. Thus, the defect in biliary GSH secretion in EHBR most likely is either at the posttranslational level of RcGshT or in the inhibition exerted by retained endogenous organic anions. In addition, there is a widespread up-regulation of GSH synthesis capacity in the tissues of EHBR.
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PMID:Alterations in glutathione homeostasis in mutant Eisai hyperbilirubinemic rats. 870 71

The conversion of gamma-glutamylcysteinylethyl ester (gamma-GCE) to glutathione in a reduced form (GSH) was examined using isolated rat hepatocytes pretreated with diethylmaleate, a GSH-depletor. Incubation of hepatocytes with 0.1 and 5.0 mM gamma-GCE (gamma-GCE-hepatocytes) over a 30-min period resulted in time-dependent increases in intracellular GSH and nonprotein-SH (NP-SH) concentrations. Hepatocytes incubated with 5.0 mM but not 0.1 mM GSH over a period of 30 min showed a time-dependent increase in intracellular GSH concentration. In the gamma-GCE-hepatocytes pretreated with bis-(p-nitrophenyl)phosphate (BNPP), a non-specific esterase inhibitor, an enhancement of intracellular GSH concentration was markedly reduced. gamma-GCE concentration in the gamma-GCE-hepatocytes with BNPP pretreatment was significantly higher than that in the cells without BNPP pretreatment, although there was no difference in the total amount of intracellular NP-SH, i.e., gamma-GCE, GSH, gamma-glutamylcysteine, cysteine ethyl ester, and cysteine between both gamma-GCE-hepatocytes. The present results indicate that gamma-GCE is transported into liver cells more easily than GSH itself, resulting in its conversion to GSH via esterase and glutathione synthetase within the cells.
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PMID:Conversion of gamma-glutamylcysteinylethyl ester to glutathione in rat hepatocytes. 878 49

Glutathione is essential for a variety of cellular functions, and is synthesized from gamma-glutamylcysteine and glycine by the action of glutathione synthase (EC 6.3.2.3). Human glutathione synthase is a dimer of two identical subunits, each composed of 474 amino acids. Little is known about the structure-function relationships of mammalian glutathione synthases and, in order to gain a greater understanding of this critical enzyme, we have probed the role of cysteine residues by chemical modification and site-directed mutagenesis. Preincubation with thiol reagents such as p-chloromercuribenzoate, N-ethylmaleimide, iodoacetate and 5,5'-dithiobis-(2-nitrobenzoate) resulted in significant inhibition of recombinant human glutathione synthase. Each subunit contains cysteine residues at positions 294, 409 and 422, and we have prepared four different mutants by replacing individual cysteine residues, or all of the cysteine residues, with alanine. The C294A and C409A cysteine mutants retained significant residual activity, indicating that these two cysteine residues are not essential for activity. In contrast, substantial decreases in enzymic activity were detected with the C422A and cysteine-free mutants. This suggests that Cys-422 may play a significant structural or functional role in human glutathione synthase.
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PMID:Identification of an essential cysteine residue in human glutathione synthase. 900 20


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