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 recognition of lysosomal enzymes by UDP-GlcNAc: lysosomal-enzyme GlcNAc-1-phosphotransferase (phosphotransferase) is mediated by a protein structure on lysosomal enzymes. It has been previously demonstrated that lysine residues are required for phosphorylation of procathepsin L and are a common feature of the site on many lysosomal proteins. In this work, the procathepsin L recognition structure was further defined by identification of the region of the protein containing the structure and the critical lysine residues involved. Removal of the cathepsin L propeptide by low pH-induced autocatalytic processing abolished phosphorylation. The addition of either the purified propeptide or a glutathione S-transferase-propeptide fusion protein to the processed protein restored phosphorylation. Mutagenesis of individual lysine residues demonstrated that two propeptide lysine residues (Lys-54 and Lys-99) were required for efficient phosphorylation of procathepsin L. By comparison of the phosphorylation rates of procathepsin L, lysine-modified procathepsin L, and the procathepsin L oligosaccharide, lysine residues were shown to account for most, if not all, of the protein-dependent interaction. On this basis, it is concluded that the proregion lysine residues are the major elements of the procathepsin L recognition site. In addition, lysine residues in cathepsin D were shown to be as important for phosphorylation as those in procathepsin L, supporting a general model of the recognition site as a specific three-dimensional arrangement of lysine residues exposed on the surface of lysosomal proteins.
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PMID:Lysine-based structure in the proregion of procathepsin L is the recognition site for mannose phosphorylation. 779 59

Glutathione transferase P1-1 (EC 2.5.1.18) is a dimeric enzyme composed of identical subunits each containing one binding site for GSH and a second for the co-substrate e.g. 1-chloro-2,4-dinitrobenzene. Steady-state kinetics are strictly hyperbolic toward both these substrates. Replacement of Cys-47 with alanine or serine decreases the affinity for GSH and triggers a positive kinetic cooperativity with respect to the substrate. Hill coefficients were 1.31 and 1.43 for the C47A and C47S mutants. C47A/C101S and C47S/C101S double mutants display lower affinity for GSH and higher Hill coefficients (1.57 and 1.56, respectively) when compared with C47A and C47S single mutants. Conversely, replacement of Cys-101 with alanine or serine does not yield any cooperativity and any marked change of kinetic parameters. Fluorometric experiments gave sigmoidal isothermic GSH binding curves for all the Cys-47 mutants, with Hill coefficients similar to that obtained by the kinetic approach. These data, together with the activation experiments performed in the presence of S-hexylglutathione, suggest that the substitution of Cys-47 yields a dimeric low-affinity enzyme which may be revealed by the lack of a peculiar electrostatic bond between the thiolate form of Cys-47 and the protonated amino group of Lys-54.
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PMID:Site-directed mutagenesis of human glutathione transferase P1-1. Mutation of Cys-47 induces a positive cooperativity in glutathione transferase P1-1. 783 86

In the human placental glutathione transferase, Cys-47 possesses, at physiological pH values, a pK alpha value of 4.2 and may exist as an ion pair with the protonated epsilon-amino group of Lys-54. Using site-directed mutagenesis we investigate spectral, kinetic, and structural properties of Cys-47 and Lys-54 mutants. The results shown indicate that the thiolate ion detected at 229 nm should be assigned exclusively to Cys-47. The contribution of Lys-54 to the activation of Cys-47 is assessed by the spectral properties of the K54A mutant enzyme. The induced cooperativity toward glutathione, as a consequence of mutation of Lys-54 to alanine, clearly parallels that observed for the Cys-47 mutant enzymes (see the preceding paper (Ricci, G., Lo Bello, M., Caccuri, A. M., Pastore, A., Nuccetelli, M., Parker, M. W., and Federici, G. (1995) J. Biol. Chem. 270, 1243-1248) and points out the importance of this electrostatic interaction in shaping the correct spatial arrangement for the binding of glutathione and in anchoring the flexible helix alpha 2. When this ion pair is disrupted, by mutation of either residue, the flexibility of this region could be greatly increased, causing helix alpha 2 to come in contact with the other subunit and generating a structural communication, which is the basis of the observed cooperativity.
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PMID:Site-directed mutagenesis of human glutathione transferase P1-1. Spectral, kinetic, and structural properties of Cys-47 and Lys-54 mutants. 783 87

Transcription factor E2F-1 has a putative consensus sequence for phosphorylation by cyclin dependent kinase (Ser-Pro-X-Lys/Arg). Therefore, we studied the phosphorylation of E2F-1 in vivo and in vitro and its biological functions. E2F-1 was prepared by immunoprecipitation with anti-E2F-1 antibody from IMR32 lysates and was effectively phosphorylated by human cyclin A-cdk2 which was expressed in insect cells using baculovirus system. GST-E2F-1 was phosphorylated by cyclin A-cdk2 more efficiently than by cyclin E-cdk2. Cyclin D1-cdk4 phosphorylated pRB but scarcely phosphorylated GST-E2F-1 or H1 histone. The 60 kd protein precipitated with anti-E2F-1 antibody was phosphorylated in vivo. Phospho-peptide mapping indicated that its cleavage profile was identical with that of E2F-1 phosphorylated by cyclin A-cdk2 in vitro. This 60 kd protein, which is likely to be E2F-1, was not phosphorylated during the G0 and early G1 phase. Phosphorylation of E2F-1 began from the S phase while phosphorylation of pRB started nearly at G1/S. The in vivo phosphorylation of E2F-1 was inhibited by butyrolactone I, a cyclin-dependent kinase inhibitor (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). The binding of E2F-1 to E2 promoter was found to be reduced by phosphorylation of E2F-1 by cyclin A-cdk2, suggesting that phosphorylation of E2F-1 may induce shut off of gene expression at the transcriptional level. These results suggest that E2F-1 is phosphorylated by cyclin A-cdk2 in the S phase in vivo as well as in vitro and that its phosphorylation by cyclin A-cdk2 may modulate its activity.
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PMID:Phosphorylation of E2F-1 by cyclin A-cdk2. 783 23

Previous studies from our laboratory have shown that aspartic acid 101 plays an important role in glutathione interaction to rat glutathione S-transferase YaYa, while tyrosine 9 is directly involved in catalysis. Based on the available structural information, site-directed mutagenesis was conducted to examine the function of arginine, lysine, glutamine, and proline residues surrounding the GSH binding pocket. Arginine mutants R13K, R15K, R20K, and R20I retained partial enzymatic activities, while R13I and R15I lost most of their activities. Kinetic studies showed a marked increase in Km toward GSH for R15I suggesting that arginine 15 contributes significantly to the binding of GSH in the active site of glutathione S-transferase YaYa. A drastic decrease in enzymatic activities for R13I suggested the importance of the charged group of arginine 13 either in maintaining the structural integrity of the enzyme or in serving a vital role in enzymatic function. Replacement of glutamine 54 and 67 with glutamic acid or asparagine resulted in decreased enzymatic activities. Moreover, an 11-, 17-, and 9-fold increase in Km values toward GSH for mutant Q54E, Q54N, and Q67N was observed, respectively. These results suggested that glutamine 54 and 67 also contributed significantly to the binding of GSH. Proline at position 56 appears to be important for maintaining the structural integrity of the enzyme since mutants P56A and P56F were much less active and extremely less stable than that of the wild type enzyme. Both lysine mutants, K45R and K45I, exhibited substantially higher catalytic efficiencies toward both 1-chloro-2,4-dinitrobenzene and GSH than the wild type enzyme. Our data clearly show that lysine 45 is not an essential residue for catalysis nor for GSH binding in glutathione S-transferase YaYa.
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PMID:Site-directed mutagenesis of glutathione S-transferase YaYa. Mapping the glutathione-binding site. 822 40

Limited proteolysis experiments have been carried out on human placental glutathione transferase in its different forms. The reduced enzyme, as well as the oxidized form and that inactivated with cystamine were all sensitive to 10% (w/w) trypsin, under nondenaturing conditions. The proteolytic cleavage was accompanied by a concomitant loss of enzymatic activity. On the contrary, the presence of glutathione or glutathione conjugates strongly protected the reduced enzyme against inactivation and from the proteolytic attack. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and peptide sequence analysis showed that only the peptide bond between Lys 44 and Ala 45 was cleaved. Since Lys 44 has been demonstrated to be involved in the glutathione binding, it is suggested that the region surrounding this amino acid residue (alpha B helix) could be more exposed to the solvent, in the absence of glutathione. Crystallographic data also indicated that this region is flexible, supporting the idea that it may be involved in the observed conformational change upon glutathione binding.
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PMID:Conformational states of human placental glutathione transferase as probed by limited proteolysis. 834 64

Cys-47, the most reactive cysteine in the homodimeric glutathione transferase (EC 2.5.1.18) from human placenta (class Pi), displays peculiar acid base and spectroscopic properties. The thiolate form of this residue is characterized by a sharp UV absorption spectrum centered at 229 nm with an epsilon = 7,500 M-1 cm-1. The dependence of the apparent extinction coefficient on pH indicates that the sulfhydryl group of Cys-47 has a pKa value of 4.2. Moreover the dependence of the reactivity of Cys-47 toward bromopyruvate and iodoacetamide with pH resembles that found for the functional sulfhydryls of thiol proteases, which have very low pKa values and exist mainly as a mercaptide-imidazole ion pair. The apparent pKa value for Cys-47, calculated by this kinetic approach, is in good agreement with that determined spectroscopically. X-ray crystallographic data indicate that the protonated amino group of Lys-54, 4.9 A from the sulfur atom, is probably involved in the deprotonation of Cys-47. Calculation of the electrostatic potential on the sulfur atom of Cys-47 gives a theoretical pKa value of 3.5 for the sulfhydryl group. The simulated neutralization of Lys-54 shifts the pKa value of Cys-47 to a normal value of 9.5. These findings suggest that at physiological pH values, Cys-47 exists as the thiolate ion stabilized by an ion pair formation with the protonated amino group of Lys-54, and this probably accounts for its high reactivity.
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PMID:Peculiar spectroscopic and kinetic properties of Cys-47 in human placental glutathione transferase. Evidence for an atypical thiolate ion pair near the active site. 836 Jan 90

The barley stripe mosaic virus (BSMV) gamma-b gene encodes a 17 kDa cysteine-rich protein known to affect virulence and to have a role in regulating viral gene expression. We have constructed recombinant gamma-b-glutathione S-transferase fusion proteins in Escherichia coli and have determined the ability of the purified fusion proteins and various mutant derivatives to bind nucleic acids in vitro. Gel-shift analyses revealed that the wild-type gamma-b-fusion protein is able to bind RNA cooperatively. The binding affinity is highly selective for single-stranded RNA because double-stranded RNA, single-stranded and double-stranded DNA, and transfer RNA were unable to compete for binding with the labelled RNA probes. However, BSMV-specific sequence binding was not observed since a chloroplast RNA competed for binding with 32P-labelled transcripts derived from the BSMV genome. The first 44 amino acids of the 152 amino acid gamma-b fusion protein encompassing one of two cysteine-rich 'zinc finger-like' motifs, and a basic region separating the finger-like motifs are required for RNA binding. Site- specific amino acid substitutions within two groups of lysine and arginine residues located in the basic motif reduced the binding affinity of the fusion protein greatly, but cysteine and histidine substitutions designed to disrupt the finger-like motifs failed to have appreciable effects on binding. These findings indicate that the regulatory properties of gamma-b may be mediated in part by RNA binding activities.
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PMID:RNA-binding activities of barley stripe mosaic virus gamma b fusion proteins. 860 84

Cytosolic glutathione S-transferases (GSTs) from rat kidneys were purified by a combination of glutathione and S-hexylglutathione affinity columns. The isolated GSTs were subjected to reverse-phase HPLC and electrospray MS analysis. The major GST isoenzymes expressed in kidney are subunits 1, 2, 7 and 8. GST 1',3 and 4 are expressed in minor amounts. GST 10 is barely detectable in the male kidney cytosol. The molecular masses of these rat kidney GST subunits were determined by MS. The values obtained for subunits 1', 2, 3, 4, 7, 8 and 10 are identical with those obtained for rat liver GSTs. Rat kidney GST 1 consists of three polypeptides, with molecular masses of 25517, 25372 and 24982 Da. Results from peptide mapping, MS and amino-acid-sequencing analyses indicate that the major components were generated by deleting the C-terminal phenylalanine (24982 Da) and the C-terminal IFKF tetrapeptide (25372 Da) from the GST 1 subunit, respectively. The 1-chloro-2,4-dinitrobenzene-conjugating and peroxidase activities of kidney GST 1 are substantially lower than for its counterpart from rat liver. In addition, rat kidney GST 1 has an arginine and a valine residue at positions 151 and 207 respectively. The results are in contradiction with the SWISS-PROT and GenBank rat liver GST 1 cDNA-sequencing data, which give a lysine and a methionine at the corresponding positions. Further analyses indicate that rat liver GST 1 also has a C-terminal phenylalanine deletion, and an arginine and a valine residue at positions 151 and 207 respectively. However, the C-terminal-tetrapeptide-deleted form was not observed for rat liver GST 1.
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PMID:Rat kidney glutathione S-transferase 1 subunits have C-terminal truncations. 861 53

LIM domains, Cys-rich motifs containing approximately 50 amino acids found in a variety of proteins, are proposed to direct protein*protein interactions. To identify structural targets recognized by LIM domains, we have utilized random peptide library selection, the yeast two-hybrid system, and glutathione S-transferase fusions. Enigma contains three LIM domains within its carboxyl terminus and LIM3 of Enigma specifically recognizes active but not mutant endocytic codes of the insulin receptor (InsR) (Wu, R. Y., and Gill, G. N. (1994) J. Biol. Chem. 269, 25085-25090). Interaction of two random peptide libraries with glutathione S-transferase-LIM3 of Enigma indicated specific binding to Gly-Pro-Hyd-Gly-Pro-Hyd-Tyr-Ala corresponding to the major endocytic code of InsR. Peptide competition demonstrated that both Pro and Tyr residues were required for specific interaction of InsR with Enigma. In contrast to LIM3 of Enigma binding to InsR, LIM2 of Enigma associated specifically with the receptor tyrosine kinase, Ret. Ret was specific for LIM2 of Enigma and did not bind other LIM domains tested. Mutational analysis indicated that the residues responsible for binding to Enigma were localized to the carboxyl-terminal 61 amino acids of Ret. A peptide corresponding to the carboxyl-terminal 20 amino acids of Ret dissociated Enigma and Ret complexes, while a mutant that changed Asn-Lys-Leu-Tyr in the peptide to Ala-Lys-Leu-Ala or a peptide corresponding to exon16 of InsR failed to disrupt the complexes, indicating the Asn-Lys-Leu-Tyr sequence of Ret is essential to the recognition motif for LIM2 of Enigma. We conclude that LIM domains of Enigma recognize tyrosine-containing motifs with specificity residing in both the LIM domains and in the target structures.
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PMID:Specificity of LIM domain interactions with receptor tyrosine kinases. 866 33


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