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)

The function of the flexible loop which is disordered in crystal structure analysis of glutathione synthetase from Escherichia coli B has been investigated by limited proteolysis and kinetic measurements for the wild-type and mutant enzymes. Proteolysis of the intact enzyme using arginyl endopeptidase or trypsin brought about a time-dependent decrease in the enzymatic activity and the production of protein fragments. SDS-polyacrylamide gel electrophoresis and peptide sequence analysis showed that only a peptide bond between arginine 233 and glycine 234 in the loop was cleaved. Further, native polyacrylamide gel electrophoresis revealed that the cleaved enzyme retained almost the same quaternary structure as that of the wild-type enzyme. Upon protease treatment, the presence of substrates, ATP and/or gamma-L-glutamyl-L-cysteine (gamma-Glu-Cys), protected the loop from cleavage, but the presence of glycine was not capable of protecting it. In addition, replacement of arginine 233 in the loop with lysine by site-directed mutagenesis increased the Michaelis constants for gamma-Glu-Cys and glycine by factors of 28 and 213, respectively. The protection against cleavage on a similar protease incubation of this mutant enzyme was also observed in the presence of ATP and/or gamma-Glu-Cys, but the effect in the presence of both substrates was half as large as that for the wild-type enzyme. These results suggest that the loop covers the active site while ATP and gamma-Glu-Cys bind there and that it protects the unstable gamma-Glu-Cys phosphate intermediate from decomposition by bulk water.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mutational and proteolytic studies on a flexible loop in glutathione synthetase from Escherichia coli B: the loop and arginine 233 are critical for the catalytic reaction. 154 May 81

New methods for the estimation of red cell gamma-glutamylcysteine synthetase and glutathione synthetase have been developed. gamma-32P ATP is allowed to equilibrate until the gamma and beta phosphate groups are equally labelled. The amount of 32Pi released in the presence of glutamic acid and cysteine, the substrates for GC-S or in the presence of gamma-glutamylcysteine and glycine, the substrates of GSH-S, is measured. This is accomplished by extraction of the phosphomolybdate complex into isobutanol-benzene. The methods are linear with time and hemolysate concentration. Normal values are presented.
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PMID:Improved assay of the enzymes of glutathione synthesis: gamma-glutamylcysteine synthetase and glutathione synthetase. 287 3

Cumene hydroperoxide (Chp) and 4-hydroxynonenal (HNE) were used to investigate the effect of peroxidative challenge upon the glutathione (GSH) metabolism of human skin fibroblasts. Cellular GSH contents decreased during short-term incubations with Chp and oxidised glutathione (GSSG) was formed concomitantly. During longer incubations the GSH level was restored and the substrate flux through the pentose phosphate shunt increased. So in the presence of hydroperoxides the GSH level is maintained by reduction of GSSG. HNE caused a strong decrease in cellular GSH contents. Prolonged incubation with HNE lead to a rise in GSH contents above the basal level. The flux through the pentose phosphate shunt did not change during exposure to HNE. Hence, during incubation with HNE the cell maintains its GSH content by de novo synthesis of GSH. This conclusion is further substantiated by the findings with a cell strain deficient in GSH synthetase. These cells survived if incubated with Chp but not if exposed to HNE. GSH contents of normal cells from phase II (young) cultures and from phase III (aged) cultures responded similarly to Chp during short-term incubations and during a week of culture with the test compound. The flux through the pentose phosphate shunt rose much more in phase III than in phase II cells when incubated with the same concentration series of Chp. We conclude that during in vitro ageing the amount of NADPH needed to maintain cellular GSH levels in the presence of hydroperoxides increases, while the capacity to respond to such a challenge is not affected.
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PMID:Influence of cumene hydroperoxide and 4-hydroxynonenal on the glutathione metabolism during in vitro ageing of human skin fibroblasts. 380 87

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

The loop from Ile-226 to Arg-241 in the glutathione synthetase (GSHase) from Escherichia coli B is rich in glycine and alanine and too flexible to take a fixed conformation [Yamaguchi, H., Kato, H., Hata, Y., Nishioka, T., Kimura, A., Oda, J., & Katsube, Y. (1993) J. Mol. Biol. 229, 1083-1100; Tanaka, T., Kato, H., Nishioka, T., & Oda, J. (1992) Biochemistry 31, 2259-2265]. To restrict the flexibility, three residues in the loop, Pro-227, Gly-229, and Gly-240, were replaced with alanine and valine residues. Variability in conformations of the mutant loops and shifts in the distribution of conformers between the open and closed states were assessed by steady-state kinetics, X-ray crystallographic structure analysis, and proteolysis with arginyl endopeptidase. Mutant enzymes replaced with a valine residue at the basal positions of the loop (P227V, G240V, and P227V/G240V) were identical with the wild-type enzyme in their crystal structures, except the loop region. The mutant loops retained apparent conformational variability, so as to take the open and closed states and to protect the acyl phosphate intermediate from the decomposition uncoupled from glutathione synthesis, but lost the catalytic activity; Kmapp values for glycine and gamma-Glu-Cys were sensitive to the mutations and drastically increased, and the k0app value was fatally reduced in the P227V/G240V mutant enzyme. The present results suggest that adjustability of the loop to the closed state is required for the recognition of the substrates, gamma-Glu-Cys and glycine, and for the chemical interactions with the bound substrates.
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PMID:Flexibility impaired by mutations revealed the multifunctional roles of the loop in glutathione synthetase. 824 Nov 29

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

The crystal structure of glutathione synthetase from Escherichia coli B complexed with ADP, glutathione, and sulfate has been determined at 2.0 A resolution. Concerning the chemical similarity of sulfate and phosphate, this quaternary complex structure represents a pseudo enzyme-substrate complex in the reverse reaction and consequently allows us to understand the active site architecture of the E. coli glutathione synthetase. Two Mg2+ ions are coordinated with oxygen atoms from the alpha- and beta-phosphate groups of ADP and from the sulfate ion. The flexible loops, invisible in the unliganded or the binary and ternary complex structures, are fixed in the quaternary complex. The larger flexible loop (Ile226-Arg241) includes one turn of a 310-helix that comprises the binding site of the glycine moiety of GSH. The small loop (Gly164-Gly167) is involved in nucleotide binding and acts as a phosphate gripper. The side chains of Arg210 and Arg225 interact with the sulfate ion and the beta-phosphate moiety of ADP. Arg 210 is likely to interact with the carboxylate of the C-terminal gamma-glutamylcysteine in the substrate-binding form of the forward reaction. Other positively charged residues in the active site (Lys125 and Lys160) are involved in nucleotide binding, directing the phosphate groups to the right position for catalysis. Functional aspects of the active site architecture in the substrate-binding form are discussed.
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PMID:A pseudo-michaelis quaternary complex in the reverse reaction of a ligase: structure of Escherichia coli B glutathione synthetase complexed with ADP, glutathione, and sulfate at 2.0 A resolution. 881 Sep 1

The role of endogenous reduced glutathione (GSH) in tert-butyl hydroperoxide (TBHP)-induced cell injury was examined in isolated rat hepatocytes. When liver cell injury was estimated from release of transaminases from hepatocytes into the incubation medium, cell injury in hepatocytes (2 x 10(6) cells/ml) incubated in Hanks' balanced salt solution (pH 7.2) containing 1.0 mM TBHP at 37 degrees C was potentiated with enhanced lipid peroxidation by prior depletion of intracellular GSH which was induced by diethylmaleate, a GSH depletor. GSH-depleted hepatocytes were incubated with gamma-glutamylcysteinylethyl ester (gamma-ECOEt), which is known to be converted to GSH via glutathione synthetase after its hydrolysis by esterase, at concentrations of 1.0 to 10 mM in order to replenish intracellular GSH. Although TBHP-induced cell injury and lipid peroxidation were enhanced in GSH-depleted hepatocytes, these enhancements were prevented with the consumption of intracellular GSH in GSH-depleted hepatocytes pretreated with 5.0 mM gamma-ECOEt. These preventive effects were observed at any time point during the TBHP treatment over a 60 min period and depended on the concentration of gamma-ECOEt used. But, no preventive effect was found in GSH-depleted hepatocytes pretreated with 5.0 mM GSH. No prevention of the potentiation of TBHP-induced cell injury found in GSH-depleted hepatocytes occurred in GSH-depleted hepatocytes pretreated with both 5.0 mM gamma-ECOEt and 250 microM bis-(p-nitrophenyl)phosphate, a nonspecific esterase inhibitor. gamma-ECOEt treatment caused an increase in intracellular GSH content in GSH-depleted hepatocytes, while treatments of both gamma-ECOEt and the esterase inhibitor caused no increase in intracellular GSH content in the cells. These results indicate that endogenous GSH modulates TBHP-induced cell injury and lipid peroxidation in isolated rat hepatocytes. The present results suggest that endogenous GSH should play a critical role in TBHP-induced cell injury in isolated rat hepatocytes and that in rat hepatocytes treated with TBHP, enhanced lipid peroxidation with the consumption of intracellular GSH could be associated with the initiation of cell injury.
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PMID:Modulating role of endogenous reduced glutathione in tert-butyl hydroperoxide-induced cell injury in isolated rat hepatocytes. 921 82

Carbamoyl phosphate synthetase (CPS) catalyzes the production of carbamoyl phosphate which is subsequently employed in the metabolic pathways responsible for the synthesis of pyrimidine nucleotides or arginine. The catalytic mechanism of the enzyme occurs through three highly reactive intermediates: carboxyphosphate, ammonia, and carbamate. As isolated from Escherichia coli, CPS is an alpha, beta-heterodimeric protein with its three active sites separated by nearly 100 A. In addition, there are separate binding sites for the allosteric regulators, ornithine, and UMP. Given the sizable distances between the three active sites and the allosteric-binding pockets, it has been postulated that domain movements play key roles for intramolecular communication. Here we describe the structure of CPS from E. coli where, indeed, such a domain movement has occurred in response to nucleotide binding. Specifically, the protein was crystallized in the presence of a nonhydrolyzable analogue, AMPPNP, and its structure determined to 2.1 A resolution by X-ray crystallographic analysis. The B-domain of the carbamoyl phosphate synthetic component of the large subunit closes down over the active-site pocket such that some atoms move by more than 7 A relative to that observed in the original structure. The trigger for this movement resides in the hydrogen-bonding interactions between two backbone amide groups (Gly 721 and Gly 722) and the beta- and gamma-phosphate groups of the nucleotide triphosphate. Gly 721 and Gly 722 are located in a Type III' reverse turn, and this type of secondary structural motif is also observed in D-alanine:D-alanine ligase and glutathione synthetase, both of which belong to the "ATP-grasp" superfamily of proteins. Details concerning the geometries of the two active sites contained within the large subunit of CPS are described.
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PMID:Carbamoyl phosphate synthetase: closure of the B-domain as a result of nucleotide binding. 1002 28

In order to search for a common structural motif in the phosphate-binding sites of protein-mononucleotide complexes, we investigated the structural variety of phosphate-binding schemes by an all-against-all comparison of 491 binding sites found in the Protein Data Bank. We found four frequently occurring structural motifs composed of protein atoms interacting with phosphate groups, each of which appears in different protein superfamilies with different folds. The most frequently occurring motif, which we call the structural P-loop, is shared by 13 superfamilies and is characterized by a four-residue fragment, GXXX, interacting with a phosphate group through the backbone atoms. Various sequence motifs, including Walker's A motif or the P-loop, turn out to be a structural P-loop found in a few specific superfamilies. The other three motifs are found in pairs of superfamilies: protein kinase and glutathione synthetase ATPase domain like, actin-like ATPase domain and nucleotidyltransferase, and FMN-linked oxidoreductase and PRTase.
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PMID:Structural motif of phosphate-binding site common to various protein superfamilies: all-against-all structural comparison of protein-mononucleotide complexes. 1006 5


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