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)

The mitochondrial outer membrane enzyme kynurenine 3-hydroxylase (K3H) is an NADPH-dependent flavin mono-oxygenase involved in the tryptophan pathway, where it catalyzes the hydroxylation of kynurenine. K3H was transiently expressed in COS-1 cells as a glutathione S-transferase (GST) fusion protein, and the pure recombinant protein (rec-K3H) was obtained with a specific activity of about 2000 nmol.min-1.mg-1. Rec-K3H was shown to have an optimum pH at 7.5, to use NADPH more efficiently than NADH, and to contain one molecule of non-covalently bound FAD per molecule of enzyme. The mechanism of the rec-K3H-catalyzed reaction was investigated by overall initial-rate measurements, and a random mechanism in which combination of the enzyme with one substrate does not influence its affinity for the other is proposed. Further kinetic studies revealed that K3H activity was inhibited by both pyridoxal phosphate and Cl-, and that NADPH-catalyzed oxidation occurred even in the absence of kynurenine if 3-hydroxykynurenine was present, suggesting an uncoupling effect of 3-hydroxykynurenine with peroxide formation. This observation could be of clinical interest, as peroxide formation could explain the neurotoxicity of 3-hydroxykynurenine in vivo.
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PMID:Functional characterization and mechanism of action of recombinant human kynurenine 3-hydroxylase. 1067 18

We have shown previously that the solvent-induced equilibrium unfolding mechanism of class Sigma glutathione S-transferase (GST) is strongly affected by ionic strength [Stevens, Hornby, Armstrong and Dirr (1998) Biochemistry 37, 15534-15541]. The protein is dimeric and has a hydrophilic subunit interface. Here we show that ionic strength alone has significant effects on the conformation of the protein, in particular at the active site. With the use of NaCl at up to 2 M under equilibrium conditions, the protein lost 60% of its catalytic activity and the single tryptophan residue per subunit became partly exposed. The effect was independent of protein concentration, eliminating the dissociation of the dimer as a possibility for the conformational changes. This was confirmed by size-exclusion HPLC. There was no significant change in the secondary structure of the protein according to far-UV CD data. Manual-mixing and stopped-flow kinetics experiments showed a slow single-exponential salt-induced change in protein fluorescence. For equilibrium and kinetics experiments, the addition of an active-site ligand (S-hexylglutathione) completely protected the protein from the ionic-strength-induced conformational changes. This suggests that the change occurs at or near the active site. Possible structural reasons for these novel effects are proposed, such as the flexibility of the alpha-helix 2 region as well as the hydrophilic subunit interface, highlighting the importance of electrostatic interactions in maintaining the structure of the active site of this GST.
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PMID:Electrostatic interactions affecting the active site of class sigma glutathione S-transferase. 1072 18

AN9 is a glutathione S-transferase from petunia (Petunia hybrida) required for efficient anthocyanin export from the site of synthesis in the cytoplasm into permanent storage in the vacuole. For many xenobiotics it is well established that a covalent glutathione (GSH) tag mediates recognition of molecules destined for vacuolar sequestration by a tonoplast-localized ATP-binding cassette pump. Here we inquired whether AN9 catalyzes the formation of GSH conjugates with flavonoid substrates. Using high-performance liquid chromatography analysis of reaction mixtures containing enzyme, GSH, and flavonoids, including anthocyanins, we could detect neither conjugates nor a decrease in the free thiol concentration. These results suggest that no conjugate is formed in vitro. However, AN9 was shown to bind flavonoids using three assays: inhibition of the glutathione S-transferase activity of AN9 toward the common substrate 1-chloro 2,4-dinitrobenzene, equilibrium dialysis, and tryptophan quenching. We conclude that AN9 is a flavonoid-binding protein, and propose that in vivo it serves as a cytoplasmic flavonoid carrier protein.
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PMID:AN9, a petunia glutathione S-transferase required for anthocyanin sequestration, is a flavonoid-binding protein. 1093 72

(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a lipid-requiring mitochondrial enzyme that has a specific requirement of phosphatidylcholine (PC) for function. The C-terminal domain (CTBDH) of human heart BDH (residues 195-297) has now been expressed in Escherichia coli as a chimera with a soluble protein, glutathione S-transferase (GST), yielding GST-CTBDH, a novel fusion protein that has been purified and shown to selectively bind to PC vesicles. Both recombinant human heart BDH (HH-Histag-BDH) and GST-CTBDH (but not GST) form well-defined protein-lipid complexes with either PC or phosphatidylethanolamine (PE)/diphosphatidylglycerol (DPG) vesicles (but not with digalactosyl diglyceride vesicles) as demonstrated by flotation in sucrose gradients. The protein-PC complexes are stable to 0.5 M NaCl, but complexes of either HH-Histag-BDH or GST-CTBDH with PE/DPG vesicles are dissociated by salt treatment. Thrombin cleavage of GST-CTBDH, either before or after reconstitution with PC vesicles, yields CTBDH (12 111 Da by MALDI mass spectrometry) which retains lipid binding without attached GST. The BDH activator, 1-palmitoyl-2-(1-pyrenyl)decanoyl-PC (pyrenyl-PC), at <2.5% of total phospholipid in vesicles, efficiently quenches a fraction (0.36 and 0.47, respectively) of the tryptophan fluorescence of both HH-Histag-BDH and GST-CTBDH with effective Stern-Volmer quenching constants, (K(Q))(eff), of 11 and 9.3 (%)(-)(1), respectively (half-maximal quenching at approximately 0.1% pyrenyl-PC). Maximal quenching by pyrenyl-PC obtains at approximately stoichiometric pyrenyl-PC to protein ratios, reflecting high-affinity interaction of pyrenyl-PC with both HH-Histag-BDH and GST-CTBDH. The analogous pyrenyl-PE effects a similar maximal quenching of tryptophan fluorescence for both proteins but with approximately 15-fold lower (K(Q))(eff) (half-maximal quenching at approximately 1.5% pyrenyl-PE) referable to nonspecific interaction of pyrenyl-PE with HH-Histag-BDH or GST-CTBDH. Thus, the 103-residue CTBDH constitutes a PC-selective lipid binding domain of the PC-requiring BDH.
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PMID:(R)-3-hydroxybutyrate dehydrogenase: selective phosphatidylcholine binding by the C-terminal domain. 1100 6

The conformational stabilities of two homodimeric class mu glutathione transferases (GSTM1-1 and GSTM2-2) were studied by urea- and guanidinium chloride-induced denaturation. Unfolding is reversible and structural changes were followed with far-ultraviolet circular dichroism, tryptophan fluorescence, enzyme activity, chemical cross-linking, and size-exclusion chromatography. Disruption of secondary structure occurs as a monophasic transition and is independent of protein concentration. Changes in tertiary structure occur as two transitions; the first is protein concentration dependent, while the second is weakly dependent (GSTM1-1) or independent (GSTM2-2). The second transition corresponds with the secondary structure transition. Loss in catalytic activity occurs as two transitions for GSTM1-1 and as one transition for GSTM2-2. These transitions are dependent upon protein concentration. The first deactivation transition coincides with the first tertiary structure transition. Dimer dissociation occurs prior to disruption of secondary structure. The data suggest that the equilibrium unfolding/refolding of the class mu glutathione transferases M1-1 and M2-2 proceed via a three-state process: N(2) <--> 2I <--> 2U. Although GSTM1-1 and GSTM2-2 are homologous (78% identity/94% homology), their N(2) tertiary structures are not identical. Dissociation of the GSTM1-1 dimer to structured monomers (I) occurs at lower denaturant concentrations than for GSTM2-2. The monomeric intermediate for GSTM1-1 is, however, more stable than the intermediate for GSTM2-2. The intermediates are catalytically inactive and display nativelike secondary structure. Guanidinium chloride-induced denaturation yields monomeric intermediates, which have a more loosely packed tertiary structure displaying enhanced solvent exposure of its tryptophans and enhanced ANS binding. The three-state model for the class mu enzymes is in contrast to the equilibrium two-state models previously proposed for representatives of classes alpha/pi/Sj26 GSTs. Class mu subunits appear to be intrinsically more stable than those of the other GST classes.
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PMID:Equilibrium folding of dimeric class mu glutathione transferases involves a stable monomeric intermediate. 1101 13

Dictyostelium myosin II heavy chain kinase A (MHCK A), MHCK B, and MHCK C contain a novel type of protein kinase catalytic domain that displays no sequence identity to the catalytic domain present in conventional serine, threonine, and/or tyrosine protein kinases. Several proteins, including myelin basic protein, myosin regulatory light chain, caldesmon, and casein were phosphorylated by the bacterially expressed MHCK A, MHCK B, and MHCK C catalytic domains. Phosphoamino acid analyses of the proteins showed that 91 to 99% of the phosphate was incorporated into threonine with the remainder into serine. Acceptor amino acid specificity was further examined using a synthetic peptide library (MAXXXX(S/T)XXXXAKKK; where X is any amino acid except cysteine, tryptophan, serine, and threonine and position 7 contains serine and threonine in a 1.7:1 ratio). Phosphorylation of the peptide library with the three MHCK catalytic domains resulted in 97 to 99% of the phosphate being incorporated into threonine, while phosphorylation with a conventional serine/threonine protein kinase, the p21-activated kinase, resulted in 80% of the phosphate being incorporated into serine. The acceptor amino acid specificity of MHCK A was tested directly by substituting serine for threonine in a synthetic peptide and a glutathione S-transferase fusion peptide substrate. The serine-containing substrates were phosphorylated at a 25-fold lower rate than the threonine-containing substrates. The results indicate that the MHCKs are specific for the phosphorylation of threonine.
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PMID:Specific phosphorylation of threonine by the Dictyostelium myosin II heavy chain kinase family. 1127 93

Kinetic evidence suggests an acidic region in nardilysin binds polyamines and acts as a regulatory domain. The binding of approximately 5 mol of spermine/mol of nardilysin was demonstrated. The binding curve was sigmoidal exhibiting an IC(50) of approximately 118 microM and a Hill coefficient of 1.8. Spermine diminished the tryptophan fluorescence of the enzyme and increased its sensitivity to protease V8. The acidic stretch from mouse and human nardilysin were expressed as glutathione transferase fusion proteins. All fusion proteins bound spermine with an IC(50) of 40 to 110 microM. The mouse fusion protein bound approximately 7 mol of spermine exhibiting a sigmoidal binding curve and a Hill coefficient of 1.4. The human acidic stretch, containing fewer acidic residues, bound approximately 5 mol of spermine/mol with a hyperbolic binding curve. Chimeric fusion proteins containing the N-terminus of the mouse acidic region fused to the C-terminus of the human acidic region bound approximately 10 mol of spermine, while the opposite chimera bound approximately 4 mol of spermine/mol. The N-terminal region of the mouse acidic domain binds 3--4 mol spermine/mol exhibiting a Hill coefficient of 1.4, while the same region from human nardilysin binds 1 mol of spermine/mol. Spermine enhanced the sensitivity of the mouse acidic domain, but not the human acidic domain, to protease V8. Together the data support a model where the acidic stretch of nardilysin functions as an autonomous domain.
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PMID:Expression of the acidic stretch of nardilysin as a functional binding domain. 1147 15

Oxysterol-binding protein (OSBP) is 1 of 12 related proteins implicated in the regulation of vesicle transport and sterol homeostasis. A yeast two-hybrid screen using full-length OSBP as bait was undertaken to identify partner proteins that would provide clues to the function of OSBP. This resulted in the cloning of vesicle-associated membrane protein-associated protein-A (VAP-A), a syntaxin-like protein implicated in endoplasmic reticulum (ER)/Golgi vesicle transport, and phospholipid regulation in mammalian cells and yeast, respectively. By using a combination of yeast two-hybrid, glutathione S-transferase pull-down and immunoprecipitation experiments, the VAP-A-binding region in OSBP was localized to amino acids 351-442. This region did not include the pleckstrin homology (PH) domain but overlapped with the N terminus of the oxysterol binding and OSBP homology domains. C- and N-terminal truncations or deletions of VAP prevented interaction with OSBP but did not affect VAP multimerization. Although the OSBP PH domain was not necessary for VAP-A binding in vitro, interaction with VAP-A was enhanced in cells by mutation of the conserved PH domain tryptophan (OSBP W174A) or deletion of the C-terminal half of the PH domain (OSBP Delta 132-182). OSBP W174A retained oxysterol binding activity, association with phospholipid vesicles via the PH domain, and localized with VAP in unusual ER-associated structures. At 40 degrees C, misfolded ts045-vesicular stomatitis virus G protein fused to green fluorescent protein was co-localized with VAP-A/OSBP W174A structures on the ER but was exported to the Golgi when folded normally at 32 degrees C. A fluorescent ceramide analogue also accumulated in these ER inclusions, and export to the Golgi was partially inhibited as indicated by decreased Golgi staining and a 30% reduction in sphingomyelin synthesis. These studies show that OSBP binding to the ER and Golgi apparatus is regulated by its PH domain and VAP interactions, and the complex is involved at a stage of protein and ceramide transport from the ER.
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PMID:Vesicle-associated membrane protein-associated protein-A (VAP-A) interacts with the oxysterol-binding protein to modify export from the endoplasmic reticulum. 1202 75

The ancillary beta subunits modulate the activation and inactivation properties of high-voltage activated (HVA) Ca(2+) channels in an isoform-specific manner. The beta subunits bind to a high-affinity interaction site, alpha-interaction domain (AID), located in the I-II linker of HVA alpha1 subunits. Nine residues in the AID motif are absolutely conserved in all HVA channels (QQxExxLxGYxxWIxxxE), but their contribution to beta-subunit binding and modulation remains to be established in Ca(V)2.3. Mutations of W386 to either A, G, Q, R, E, F, or Y in Ca(V)2.3 disrupted [(35)S]beta3-subunit overlay binding to glutathione S-transferase fusion proteins containing the mutated I-II linker, whereas mutations (single or multiple) of nonconserved residues did not affect the protein-protein interaction with beta3. The tryptophan residue at position 386 appears to be an essential determinant as substitutions with hydrophobic (A and G), hydrophilic (Q, R, and E), or aromatic (F and Y) residues yielded the same results. beta-Subunit modulation of W386 (A, G, Q, R, E, F, and Y) and Y383 (A and S) mutants was investigated after heterologous expression in Xenopus oocytes. All mutant channels expressed large inward Ba(2+) currents with typical current-voltage properties. Nonetheless, the typical hallmarks of beta-subunit modulation, namely the increase in peak currents, the hyperpolarization of peak voltages, and the modulation of the kinetics and voltage dependence of inactivation, were eliminated in all W386 mutants, although they were preserved in part in Y383 (A and S) mutants. Altogether these results suggest that W386 is critical for beta-subunit binding and modulation of HVA Ca(2+) channels.
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PMID:A specific tryptophan in the I-II linker is a key determinant of beta-subunit binding and modulation in Ca(V)2.3 calcium channels. 1220 69

Human immunodeficiency virus 1 (HIV-1) encodes a gene product, Vpr, that facilitates the nuclear uptake of the viral pre-integration complex in non-dividing cells and causes infected cells to arrest in the G(2) phase of the cell cycle. Vpr was also shown to cause mitochondrial dysfunction in human cells and budding yeasts, an effect that was proposed to lead to growth arrest and cell killing in budding yeasts and apoptosis in human cells. In this study, we used a genetic selection in Saccharomyces cerevisiae to identify hexameric peptides that suppress the growth arrest phenotype mediated by Vpr. Fifteen selected glutathione S-transferase (GST)-fused peptides were found to overcome to different extents Vpr-mediated growth arrest. Amino acid analysis of the inhibitory peptide sequences revealed the conservation of a di-tryptophan (diW) motif. DiW-containing GST-peptides interacted with Vpr in GST pull-down assays, and their level of interaction correlated with their ability to overcome Vpr-mediated growth arrest. Importantly, Vpr-binding GST-peptides were also found to alleviate Vpr-mediated apoptosis and G(2) arrest in HIV-1-producing CD4(+) T cell lines. Furthermore, they co-localized with Vpr and interfered with its nuclear translocation. Overall, this study defines a class of diW-containing peptides that inhibit HIV-1 Vpr biological activities most likely by interacting with Vpr and interfering with critical protein interactions.
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PMID:Genetic selection of peptide inhibitors of human immunodeficiency virus type 1 Vpr. 1237 52


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