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:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recombinant plasmids containing the double-stranded cDNA sequences of mRNA for the Mr 22,000 ligandin (glutathione S-transferase B) subunit (Ya) have been constructed. The DNA sequence of an insert corresponding to the middle and 3' regions of the mRNA was determined and an amino acid sequence was proposed for the ligandin Ya subunit. The proposed sequence reveals a high content of basic amino acids (Arg and Lys) and Leu, is consistent with the amino acid composition, and predicts the correct number of peptides derived from tryptic digests reported for ligandin.
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PMID:Rat ligandin mRNA molecular cloning and sequencing. 668 42

A study of the structure of glutathione transferase B (ligandin) has been made with a view to understanding the relationship between the structures of the subunits of which it is composed. It consists of a mixture of a homodimer (YaYa) and a heterodimer (YaYc) in which the monomers are defined by their apparent molecular weights, that of Ya being 22000 and Yc 25000. Soluble tryptic peptides from the native homodimer YaYa have been compared with those from an artificial homodimer YcYc produced by rehybridization of native YaYc. Approximately 10 peptides specific to YaYa, 12 specific to YcYc and 21 common to both have been detected. Some of the above peptides are derived from variants of the monomers themselves. YaYa and YcYc have two C termini which are the same in both dimers, namely phenylalanine and lysine. Also there are four cysteinyl peptides, of which three are common to YaYa and YcYc and one specific to each. These results suggest that Ya and Yc are derived from at least two different but related genes.
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PMID:Evidence that the Ya and Yc subunits of glutathione transferase B (ligandin) are the products of separate genes. 714 Jul 37

The cytoplasmic insulin receptor substrate-1 (IRS-1), which is multiply phosphorylated in vivo on tyrosine residues, is a known binding protein for the tandem src homology 2 (SH2) domain-containing protein tyrosine phosphatase, SH-PTP2. Eleven phosphotyrosyl (pY) peptides from IRS-1 were screened for allosteric activation of SH-PTP2 phosphatase activity toward phosphorylated, reduced, carboxyamidomethylated, and maleylated-lysozyme. Peptides IRS-1pY895, IRS-1pY1172, and IRS-1pY1222 showed up to 50-fold acceleration of dephosphorylation. Analyses of Arg to Lys mutants in either or both SH2 domains indicate that both the N-terminal (N-SH2) and C-terminal (C-SH2) domains function in allosteric activation. Direct determination by surface plasmon resonance of the dissociation constants between pY peptides and glutathione S-transferase fusions to N-SH2 and C-SH2 domains reveals a 240-fold preference of the N-SH2 domain (compared with the C-SH2 domain) for IRS-1pY1172. The N-SH2 domain prefers IRS-1pY1172 > IRS-1pY895 > IRS-1pY1222, whereas C-SH2 domain prefers IRS-1pY1222 > IRS-1pY895 > IRS-1pY1172. These data suggest that each SH2 domain can bind to a distinct pY sequence of multiply phosphorylated protein substrates such as IRS-1, while activating hydrolysis at a third pY sequence bound in the SH-PTP2 active site. In addition, proteolysis and truncation studies reveal an autoregulatory function for the C-terminal region of SH-PTP2. Limited tryptic cleavage within the C-terminus results in 27-fold activation of protein tyrosine phosphatase activity. The activated tryptic fragment cannot be further activated by pY peptide binding to the SH2 domains indicating that autoregulatory functions of the SH2 domains are dependent on the C-terminal region. These data suggest that multiple levels for control of SH-PTP2 enzymatic activity may exist in vitro and in vivo.
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PMID:Activation of the SH2-containing protein tyrosine phosphatase, SH-PTP2, by phosphotyrosine-containing peptides derived from insulin receptor substrate-1. 751 3

The 3-dimensional crystal structure of glutathione S-transferase (GST) of Schistosoma japonicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV-1) (Muster T et al., 1993, J Virol 67:6642-6647) was determined at 2.5 A resolution. The structure of the 3-3 isozyme rat GST of the mu gene class (Ji X, Zhang P, Armstrong RN, Gilliland GL, 1992, Biochemistry 31:10169-10184) was used as a molecular replacement model. The structure consists of a 4-stranded beta-sheet and 3 alpha-helices in domain 1 and 5 alpha-helices in domain 2. The space group of the Sj GST crystal is P4(3)2(1)2, with unit cell dimensions of a = b = 94.7 A, and c = 58.1 A. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2-fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu-Leu-Asp-Lys-Trp-Ala) fused to the C-terminus of Sj GST forms a loop stabilized by symmetry-related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes mu, alpha, and pi. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed.
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PMID:Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV. 753 46

We determined the electrophilic substrate-binding site of rat glutathione S-transferase P (GST-P) by photoaffinity labeling using the photosensitive compound S-[2-(2-fluoro-4-nitrophenoxy)ethyl]glutathione. This photosensitive glutathione analogue inhibited the catalytic activity in a competitive manner against both glutathione and 1-chloro-2,4-dinitrobenzene, a putative electrophilic substrate. The enzyme kinetics indicated that the photoactivatable glutathione analogue was specifically bound at the active site, which consisted of glutathione-binding (G-site) and the electrophilic substrate-binding (H-site) regions. The procedure involved the following steps: S-[2-(2-fluoro-4-nitrophenoxy)ethyl]glutathione was photochemically reacted with a purified recombinant GST-P expressed in Escherichia coli using ultraviolet irradiation for 30 min on ice. After the reaction, only the GST-P complexed with the glutathione analogue was prepared with glutathione-immobilized agarose. The GST-P covalently bound with the analogue was digested with lysyl endopeptidase (Achromobacter protease I), and the peptides were separated by high-performance liquid chromatography. Only a single major peak with appreciable absorbance at 340 nm was observed by peptide mapping. The peptide was collected and analyzed using an automated peptide sequencer (ABI 477A). Amino acid sequence analysis showed that this peptide consisted of seven amino acid residues corresponding to the sequence at positions 122-128 of GST-P (Ala-Leu-Pro-Gly-Xaa-Leu-Lys). No appreciable phenylthiohydantoin-amino acid was detected at the fifth cycle, which indicated that His126 was chemically labeled with the photosensitive glutathione analogue. It was concluded that His126 was one of the amino acid residues forming the electrophilic substrate-binding site of GST-P.
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PMID:Identification of the electrophilic substrate-binding site of glutathione S-transferase P by photoaffinity labeling. 755 38

The C-terminal region of rat glutathione S-transferase P (GST-P) was deleted by either carboxypeptidase (CPase) A and B or site-specific truncation to evaluate the role of the region in the catalytic mechanism. The C-terminal sequence from the 201st to 209th amino-acid residues is Arg-Pro-Ile-Asn-Gly-Asn-Gly-Lys-Gln. When seven of the C-terminal amino-acid residues from the C-terminus were removed by the CPases, the catalytic activity decreased in parallel with the amino-acid removal, amounting to less than 5% of that of the wild-type GST-P. On the other hand, a decrease of the catalytic activity was observed in a different manner when the C-terminal sequence was site-specifically truncated. The VmaxGSH/KmGSH values of the mutants withthree (GSTd207-209), four (GSTd206-209) and seven (GSTd203-209) C-terminal amino-acid residues deleted, were comparable or similar to that of the wild-type GST-P, whereas those of five (GSTd205-209), six (GSTd204-209), and eight (GSTd202-209) amino-acid residue-truncated mutants decreased to 43%, 40%, and 19% of that of the wild-type GST-P, respectively. Similar results were obtained as for VmaxCDNB/KmCDNB. The nine amino-acid residue-truncated mutant showed no catalytic activity. Heat treatment at 50 degrees C for 5 min had little effect on the catalytic activities of the wild-type GST-P and GSTd204-209, whereas those of GSTd207-209, GSTd206-209, GSTd203-209 and GSTd202-209 decreased to 22%, 27%, 18% and 10%, respectively, compared to the catalytic activity of the non-treated enzymes. Considering these results, it is concluded that the C-terminal region, Arg201-Gln209, has an important role in stabilizing the active-site conformation.
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PMID:The C-terminal region, Arg201-Gln209, of glutathione S-transferase P contributes to stability of the active-site conformation. 757 28

The Ste20p protein kinase was immunopurified from yeast cells and analyzed in an in vitro assay system. Ste20p immune complexes exhibited autophosphorylating activity at serine and threonine residues and specifically phosphorylated a bacterially expressed glutathione S-transferase (GST) fusion of Ste11p (a mitogen-activated protein or extracellular signal-regulated kinase kinase (MEK) kinase homologue) at serine and threonine residues. In contrast, GST fusions either of Ste7p (a MEK homologue) or the beta-subunit of the mating response G-protein and immunoprecipitated Ste5p were not phosphorylated by the Ste20p immune complexes. Myelin basic protein was identified as an excellent in vitro substrate, whereas histone H1 was only poorly phosphorylated. Evidence was obtained that autophosphorylation might play a regulatory role for the in vitro kinase activity. The in vitro activity was found to be Ca(2+)-independent. Both the in vivo and in vitro activities were abolished by mutational changes of either the conserved lysine residue 649 within the ATP binding site or threonine 777 between the catalytic subdomains VII and VIII. Wild-type Ste20p and the catalytically inactive T777A mutant were identified as phosphoproteins in vivo. The phosphorylation occurred at serine and threonine residues independent of pheromone stimulation. Based on the genetically determined significance of Ste20p in pheromone signal transduction and on our in vitro studies, we propose the model that Ste20p represents a yeast MEK kinase kinase whose function is to link G-protein-coupled receptors through G beta gamma to a mitogen-activated protein kinase module.
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PMID:Molecular characterization of Ste20p, a potential mitogen-activated protein or extracellular signal-regulated kinase kinase (MEK) kinase kinase from Saccharomyces cerevisiae. 760 57

Mutations in the human glucokinase (GK) gene are thought to cause maturity-onset diabetes of youth (MODY) by leading to the production of enzymes with reduced catalytic activities and increased glucose Km values. However, in some cases the diabetic phenotype is more severe than might be predicted from these apparent kinetic effects alone. To determine whether these mutations might also effect other characteristics of the enzyme, nine MODY-associated mutants were expressed as fusion proteins with Schistosoma japonicum glutathione S-transferase (GST) and compared with three wild-type human GK isoforms that were also expressed in the same manner. Three GST-GK isoforms (liver 1, liver 2 and islet) were kinetically indistinguishable from each other and from purified rat liver GK. Noteworthy is a glucose-induced fit effect for the interaction of trinitrophenyl (TNP)-ATP with GST-GK, whereby glucose significantly increased the affinity of TNP-ATP binding to GST-GK without changing the stoichiometry of binding. The nine MODY-associated mutations studied either showed diminished catalytic activity, substrate affinities, allosteric regulation, or stability of the fusion enzyme. We conclude that: (1) Gly261 and Lys414 are important for ATP binding; (2) Val203 may be essential for a glucose-induced fit effect; and (3) the stability of fusion protein may be significantly reduced when Glu300 is replaced by Lys. These results suggest that, in addition to effects on the Km and Vmax. of GK, a decrease in the ATP-binding affinity or stability of the mutated enzyme may also contribute to a reduction of GK activity in individuals with GK-MODY. In the B-cell this would have the effect of blunting glucose-stimulated insulin release, thereby contributing to the diabetic phenotype.
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PMID:Variable effects of maturity-onset-diabetes-of-youth (MODY)-associated glucokinase mutations on substrate interactions and stability of the enzyme. 761 52

p44erk1 is a member of a family of tyrosyl-phosphorylated and mitogen-activated protein (MAP) kinases that participate in cell cycle control. A full-length erk1 cDNA was isolated from a human hepatoma cell line (Hep G2) library. The erk1 cDNA clone shared approximately 96% predicted amino acid identity with partial sequences of rodent erk1 cognates, and the erk1 gene was assigned to human chromosome 16 by hybrid panel analysis. Human erk1 expressed in Escherichia coli as a glutathione S-transferase fusion (GST-Erk1) protein was substantially phosphorylated on tyrosine in vivo. It underwent further autophosphorylation in vitro (up to 0.01 mol of P per mol) at the regulatory Tyr-204 site and at additional tyrosine and serine residues. Threonine autophosphorylation, presumably at the regulatory Thr-202 site, was also detected weakly when the recombinant kinase was incubated in the presence of manganese, but not in the presence of magnesium. Before and after cleavage of the GST-Erk1 protein with thrombin, it exhibited a relatively high level of myelin basic protein phosphotransferase activity, which could be reduced eightfold by treatment of the kinase with the protein-tyrosine phosphatase CD45, but not by treatment with the protein-serine/threonine phosphatase 2A. The protein-tyrosine kinase p56lck catalyzed phosphorylation of GST-Erk1 at two autophosphorylations sites, including Tyr-204, and at a novel site. A further fivefold stimulation of the myelin basic protein phosphotransferase activity of the GST-Erk1 was achieved in the presence of a partially purified MAP kinase kinase from sheep platelets. Under these circumstances, there was primarily an enhancement of the tyrosine phosphorylation of GST-Erk1. This MAP kinase kinase also similarly phosphorylated a catalytically compromised version of GST-Erk1 in which Lys-71 was converted to Ala by site-directed mutagenesis.
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PMID:Molecular cloning, expression, and characterization of the human mitogen-activated protein kinase p44erk1. 768 43

Arg15 is a conserved active-site residue in class Alpha glutathione transferases. X-ray diffraction studies of human glutathione transferase A1-1 have shown that N epsilon of this amino acid residue is adjacent to the sulfur atom of a glutathione derivative bound to the active site, suggesting the presence of a hydrogen bond. The phenolic hydroxyl group of Tyr9 also forms a hydrogen bond to the sulfur atom of glutathione, and removal of this hydroxyl group causes partial inactivation of the enzyme. The present study demonstrates by use of site-directed mutagenesis the functional significance of Arg15 for catalysis. Mutation of Arg15 into Leu reduced the catalytic activity by 25-fold, whereas substitution by Lys caused only a threefold decrease, indicating the significance of a positively charged residue at position 15. Mutation of Arg15 into Ala or His caused a substantial reduction of the specific activity (200 or 400-fold, respectively), one order of magnitude more pronounced than the effect of the Tyr9-->Phe mutation. Double mutations involving residues 9 and 15 demonstrated that the effects of mutations at the two positions were additive except for the substitution of His for Arg15, which appeared to cause secondary structural effects. The pKa value of the phenolic hydroxyl of Tyr9 was determined by UV absorption difference spectroscopy and was found to be 8.1 in the wild-type enzyme. The corresponding pKa values of mutants R15K, R15H and R15L were 8.5, 8.7 and 8.8, respectively, demonstrating the contribution of the guanidinium group of Arg15 to the electrostatic field in the active site. Addition of glutathione caused an increased pKa value of Tyr9; this effect was not obtained with S-methylglutathione. These results show that Tyr9 is protonated when glutathione is bound to the enzyme at physiological pH values. The involvement of an Arg residue in the binding and activation of glutathione is a feature that distinguishes class Alpha glutathione transferases from members in other glutathione transferase classes.
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PMID:Functional significance of arginine 15 in the active site of human class alpha glutathione transferase A1-1. 772 30


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