scholarly journals Photoaffinity labelling of the active site of the rat glutathione transferases 3-3 and 1-1 and human glutathione transferase A1-1

1994 ◽  
Vol 302 (2) ◽  
pp. 383-390 ◽  
Author(s):  
R J Cooke ◽  
R Björnestedt ◽  
K T Douglas ◽  
J H McKie ◽  
M D King ◽  
...  
Keyword(s):  
Rat Liver ◽  
Active Site ◽  
Glutathione Transferase ◽  
Competitive Inhibitor ◽  
Glutathione Transferases ◽  
Molecular Graphics ◽  
Hydrophobic Region ◽  
Preparative Scale ◽  
Photoaffinity Labelling ◽  
Wide Range

The glutathione transferases (GSTs) form a group of enzymes responsible for a wide range of molecular detoxications. The photoaffinity label S-(2-nitro-4-azidophenyl)glutathione was used to study the hydrophobic region of the active site of the rat liver GST 1-1 and 2-2 isoenzymes (class Alpha) as well as the rat class-Mu GST 3-3. Photoaffinity labelling was carried out using a version of S-(2-nitro-4-azidophenyl)glutathione tritiated in the arylazido ring. The labelling occurred with higher levels of radioisotope incorporation for the Mu than the Alpha families. Taking rat GST 3-3, 1.18 (+/- 0.05) mol of radiolabel from S-(2-nitro-4-azidophenyl)glutathione was incorporated per mol of dimeric enzyme, which could be blocked by the presence of the strong competitive inhibitor, S-tritylglutathione (Ki = 1.4 x 10(-7) M). Radiolabelling of the protein paralleled the loss of enzyme activity. Photoaffinity labelling by tritiated S-(2-nitro-4-azidophenyl)glutathione on a preparative scale (in the presence and absence of S-tritylglutathione) followed by tryptic digestion and purification of the labelled peptides indicated that GST 3-3 was specifically photolabelled; the labelled peptides were sequenced. Similarly, preparative photoaffinity labelling by S-(2-nitro-4-azidophenyl)glutathione of the rat liver 1-1 isoenzyme, the human GST A1-1 and the human-rat chimaeric GST, H1R1/1, was carried out with subsequent sequencing of radiolabelled h.p.l.c.-purified tryptic peptides. The results were interpreted by means of molecular-graphics analysis to locate photoaffinity-labelled peptides using the X-ray-crystallographic co-ordinates of rat GST 3-3 and human GST A1-1. The molecular-graphical analysis indicated that the labelled peptides are located within the immediate vicinity of the region occupied by S-substituted glutathione derivatives bound in the active-site cavity of the GSTs investigated.

scholarly journals Chemical modification of rat liver microsomal glutathione transferase defines residues of importance for catalytic function

1990 ◽  
Vol 272 (2) ◽  
pp. 479-484 ◽  
Author(s):  
C Andersson ◽  
R Morgenstern
Keyword(s):  
Chemical Modification ◽  
Rat Liver ◽  
Active Site ◽  
Glutathione Transferase ◽  
Amino Acid Residues ◽  
Active Site Residues ◽  
Histidine Residues ◽  
Catalytic Function ◽  
Low Efficiency ◽  
Microsomal Glutathione

Amino acid residues that are essential for the activity of rat liver microsomal glutathione transferase have been identified using chemical modification with various group-selective reagents. The enzyme reconstituted into phosphatidylcholine liposomes does not require stabilization with glutathione for activity (in contrast with the purified enzyme in detergent) and can thus be used for modification of active-site residues. Protection by the product analogue and inhibitor S-hexylglutathione was used as a criterion for specificity. It was shown that the histidine-selective reagent diethylpyrocarbonate inactivated the enzyme and that S-hexylglutathione partially protected against this inactivation. All three histidine residues in microsomal glutathione transferase could be modified, albeit at different rates. Inactivation of 90% of enzyme activity was achieved within the time period required for modification of the most reactive histidine, indicating the functional importance of this residue in catalysis. The arginine-selective reagents phenylglyoxal and 2,3-butanedione inhibited the enzyme, but the latter with very low efficiency; therefore no definitive assignment of arginine as essential for the activity of microsomal glutathione transferase can be made. The amino-group-selective reagents 2,4,6-trinitrobenzenesulphonate and pyridoxal 5′-phosphate inactivated the enzyme. Thus histidine residues and amino groups are suggested to be present in the active site of the microsomal glutathione transferase.

scholarly journals Paradoxical inhibition of rat glutathione transferase 4-4 by indomethacin explained by substrate-inhibitor-enzyme complexes in a random-order sequential mechanism

1988 ◽  
Vol 250 (3) ◽  
pp. 705-711 ◽  
Author(s):  
U H Danielson ◽  
B Mannervik
Keyword(s):  
Glutathione Transferase ◽  
Random Order ◽  
Competitive Inhibitor ◽  
Glutathione Transferases ◽  
Low Concentrations ◽  
Standard Assay ◽  
Sequential Mechanism ◽  
Substrate Inhibitor ◽  
Enzyme Complexes ◽  
Assay Conditions

Under standard assay conditions, with 1-chloro-2,4-dinitrobenzene (CDNB) as electrophilic substrate, rat glutathione transferase 4-4 is strongly inhibited (I50 = 1 microM) by indomethacin. No other glutathione transferase investigated is significantly inhibited by micromolar concentrations of indomethacin. Paradoxically, the strong inhibition of glutathione transferase 4-4 was dependent on high (millimolar) concentrations of CDNB; at low concentrations of this substrate or with other substrates the effect of indomethacin on the enzyme was similar to the moderate inhibition noted for other glutathione transferases. In general, the inhibition of glutathione transferases can be explained by a random-order sequential mechanism, in which indomethacin acts as a competitive inhibitor with respect to the electrophilic substrate. In the specific case of glutathione transferase 4-4 with CDNB as substrate, indomethacin binds to enzyme-CDNB and enzyme-CDNB-GSH complexes with an even greater affinity than to the corresponding complexes lacking CDNB. Under presumed physiological conditions with low concentrations of electrophilic substrates, indomethacin is not specific for glutathione transferase 4-4 and may inhibit all forms of glutathione transferase.

scholarly journals Proposed Mechanism for Monomethylarsonate Reductase Activity of Human Omega-class Glutathione Transferase GSTO1

2021 ◽  
Author(s):  
Aaron J Oakley
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Binding Sites ◽  
Glutathione Transferase ◽  
Reductase Activity ◽  
Inorganic Arsenic ◽  
Glutathione Transferases ◽  
Public Health Issue ◽  
Major Public Health Issue ◽  
Glutathione Binding

Contamination of drinking water with toxic inorganic arsenic is a major public health issue. The mechanisms of enzymes and transporters in arsenic elimination are therefore of interest. The human omega-class glutathione transferases have been previously shown to possess monomethylarsonate (V) reductase activity. To further understanding of this activity, molecular dynamics of human GSTO1-1 bound to glutathione with a monomethylarsonate isostere were simulated to reveal putative monomethylarsonate binding sites on the enzyme. The major binding site is in the active site, adjacent to the glutathione binding site. Based on this and previously reported biochemical data, a reaction mechanism for this enzyme is proposed. Further insights were gained from comparison of the human omega-class GSTs to homologs from a range of animals.

scholarly journals Cytosolic glutathione transferases from rat liver. Primary structure of class alpha glutathione transferase 8-8 and characterization of low-abundance class Mu glutathione transferases

1989 ◽  
Vol 261 (2) ◽  
pp. 531-539 ◽  
Author(s):  
P Alin ◽  
H Jensson ◽  
E Cederlund ◽  
H Jörnvall ◽  
B Mannervik
Keyword(s):  
Rat Liver ◽  
Glutathione Transferase ◽  
Glutathione Transferases ◽  
Rat Lung ◽  
Cytosol Fraction ◽  
Methionine Residue ◽  
Isoelectric Points ◽  
Cytosolic Glutathione ◽  
Lung And Kidney

Six GSH transferases with neutral/acidic isoelectric points were purified from the cytosol fraction of rat liver. Four transferases are class Mu enzymes related to the previously characterized GSH transferases 3-3, 4-4 and 6-6, as judged by structural and enzymic properties. Two additional GSH transferases are distinguished by high specific activities with 4-hydroxyalk-2-enals, toxic products of lipid peroxidation. The most abundant of these two enzymes, GSH transferase 8-8, a class Alpha enzyme, has earlier been identified in rat lung and kidney. The amino acid sequence of subunit 8 was determined and showed a typical class Alpha GSH transferase structure including an N-acetylated N-terminal methionine residue.

scholarly journals Isoenzymes of glutathione transferase in rat kidney cytosol

1985 ◽  
Vol 230 (3) ◽  
pp. 609-615 ◽  
Author(s):  
C Guthenberg ◽  
H Jensson ◽  
L Nyström ◽  
E Österlund ◽  
M K Tahir ◽  
...  
Keyword(s):  
Rat Liver ◽  
Liver Enzymes ◽  
Glutathione Transferase ◽  
Ethacrynic Acid ◽  
Rat Kidney ◽  
Glutathione Transferases ◽  
Substrate Specificities ◽  
Additional Peak ◽  
Rat Kidney Cytosol ◽  
Rat Liver Cytosol

Glutathione transferases from rat kidney cytosol were purified about 40-fold by chromatography on S-hexylglutathione linked to epoxy-activated Sepharose 6B. Further purification by fast protein liquid chromatography with chromatofocusing in the pH interval 10.6-7.6 resolved five major peaks of activity with 1-chloro-2,4-dinitrobenzene as the second substrate. Four of the peaks were identified with rat liver transferases 1-1, 1-2, 2-2 and 4-4 respectively. The criteria used for identification included physical properties, reactions with specific antibodies, substrate specificities and sensitivities to several inhibitors. The fourth major peak is a ‘new’ form of transferase, which has not been found in rat liver. This isoenzyme, glutathione transferase 7-7, has a lower apparent subunit Mr than any of the transferases isolated from rat liver cytosol, and does not react with antibodies raised against the liver enzymes. Glutathione transferases 3-3 and 3-4, which are abundant in liver, were only present in very small amounts. In a separate chromatofocusing separation in a lower pH interval, an additional peak was eluted at pH 6.3. This isoenzyme is characterized by its high activity with ethacrynic acid.

scholarly journals Human Glutathione Transferase T2-2 Discloses Some Evolutionary Strategies for Optimization of Substrate Binding to the Active Site of Glutathione Transferases

2000 ◽  
Vol 276 (8) ◽  
pp. 5427-5431 ◽  
Author(s):  
Anna Maria Caccuri ◽  
Giovanni Antonini ◽  
Philip G. Board ◽  
Jack Flanagan ◽  
Michael W. Parker ◽  
...  
Keyword(s):  
Active Site ◽  
Glutathione Transferase ◽  
Substrate Binding ◽  
Glutathione Transferases ◽  
Evolutionary Strategies

scholarly journals Structure-activity relationships of 4-hydroxyalkenals in the conjugation catalysed by mammalian glutathione transferases

1987 ◽  
Vol 247 (3) ◽  
pp. 707-713 ◽  
Author(s):  
U H Danielson ◽  
H Esterbauer ◽  
B Mannervik
Keyword(s):  
Chain Length ◽  
Active Site ◽  
Glutathione Transferase ◽  
Physiological Role ◽  
Catalytic Efficiency ◽  
Glutathione Transferases ◽  
Side Chain ◽  
Structure Activity ◽  
Cytosolic Glutathione ◽  
Free Energy Of Binding

The substrate specificities of 15 cytosolic glutathione transferases from rat, mouse and man have been explored by use of a homologous series of 4-hydroxyalkenals, extending from 4-hydroxypentenal to 4-hydroxypentadecenal. Rat glutathione transferase 8-8 is exceptionally active with the whole range of 4-hydroxyalkenals, from C5 to C15. Rat transferase 1-1, although more than 10-fold less efficient than transferase 8-8, is the second most active transferase with the longest chain length substrates. Other enzyme forms showing high activities with these substrates are rat transferase 4-4 and human transferase mu. The specificity constants, kcat./Km, for the various enzymes have been determined with the 4-hydroxyalkenals. From these constants the incremental Gibbs free energy of binding to the enzyme has been calculated for the homologous substrates. The enzymes responded differently to changes in the length of the hydrocarbon side chain and could be divided into three groups. All glutathione transferases displayed increased binding energy in response to increased hydrophobicity of the substrate. For some of the enzymes, steric limitations of the active site appear to counteract the increase in binding strength afforded by increased chain length of the substrate. Comparison of the activities with 4-hydroxyalkenals and other activated alkenes provides information about the active-site properties of certain glutathione transferases. The results show that the ensemble of glutathione transferases in a given species may serve an important physiological role in the conjugation of the whole range of 4-hydroxyalkenals. In view of its high catalytic efficiency with all the homologues, rat glutathione transferase 8-8 appears to have evolved specifically to serve in the detoxication of these reactive compounds of oxidative metabolism.

scholarly journals Epsilon glutathione transferases possess a unique class-conserved subunit interface motif that directly interacts with glutathione in the active site

2015 ◽  
Vol 35 (6) ◽  
Author(s):  
Jantana Wongsantichon ◽  
Robert C. Robinson ◽  
Albert J. Ketterman
Keyword(s):  
Drosophila Melanogaster ◽  
Active Site ◽  
Active Sites ◽  
Glutathione Transferase ◽  
Glutathione Transferases ◽  
Conserved Motif ◽  
Subunit Interface ◽  
Dimeric Subunit

Analysis of a new structure of an Epsilon class glutathione transferase from Drosophila melanogaster reveals a highly conserved motif that spans the dimeric subunit interface and connects the two active sites.

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