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

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.

Download Full-text

Carboxymethylhydroxymuconic semialdehyde dehydrogenase in the 4-hydroxyphenylacetate catabolic pathway of Pseudomonas putida

Biochemistry and Cell Biology ◽

10.1139/o86-169 ◽

1986 ◽

Vol 64 (12) ◽

pp. 1288-1293 ◽

Cited By ~ 6

Author(s):

Josefa M. Alonso ◽

Amando Garrido-Pertierra

Keyword(s):

Pseudomonas Putida ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Competitive Inhibitor ◽

Catabolic Pathway ◽

Ph Optimum ◽

Semialdehyde Dehydrogenase ◽

Standard Assay ◽

Assay Conditions

5-Carboxymethyl-2-hydroxymuconic semialdehyde (CHMSA) dehydrogenase in the 4-hydroxyphenylacetate meta-cleavage pathway was purified from Pseudomonas putida by gel filtration, anion-exchange, and affinity chromatographies. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis analysis suggested an approximate tetrameric molecular weight of 200 000. The purified enzyme showed a pH optimum at 7.8. The temperature–activity relationship for the enzyme from 27 to 45 °C showed broken Arrhenius plots with an inflexion at 36–37 °C. Under standard assay conditions, the enzyme acted preferentially with NAD. It could also catalyze the reduction with NADP (which had a higher Km), at 18% of the rate observed for NAD. The following kinetic parameters were found: Km(NAD) = 20.0 ± 3.6 μM, Km(CHMSA) = 8.5 ± 1.8 μM, and Kd(enzyme–NAD complex) = 7.8 ± 2.0 μM. The product NADH acted as a competitive inhibitor against NAD.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj3020383 ◽

1994 ◽

Vol 302 (2) ◽

pp. 383-390 ◽

Cited By ~ 9

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.

Download Full-text

Characterization of adenosine deaminase (ADA) in Hemolymph of Biomphalaria glabrata

Brazilian Journal of Biology ◽

10.1590/s1519-69842005000200022 ◽

2005 ◽

Vol 65 (2) ◽

pp. 371-376 ◽

Cited By ~ 2

Author(s):

M. R. Vale ◽

R. V. Pereira ◽

S. M. Almeida ◽

Y. M. Almeida ◽

S. F. L. C. Nunes

Keyword(s):

Adenosine Deaminase ◽

Incubation Temperature ◽

Small Variation ◽

Healthy Human ◽

Ph Variation ◽

Cellular Events ◽

Low Concentrations ◽

Key Enzyme ◽

Assay Conditions

Adenosine is an important signaling molecule for many cellular events. Adenosine deaminase (ADA) is a key enzyme for the control of extra- and intra-cellular levels of adenosine. Activity of ADA was detected in hemolymph of B. glabrata and its optimum assay conditions were determined experimentally. The pH variation from 6.2 to 7.8 caused no significant change in ADA activity. Using adenosine as a substrate, the apparent Km at pH 6.8 was 734 µmols.L-1. Highest activity was found at 37ºC. Standard assay conditions were established as being 15 minutes of incubation time, 0.4 µL of pure hemolymph per assay, pH 6.8, and 37ºC. This enzyme showed activities of 834 ± 67 µmol.min-1.L-1 (25ºC) and 2029 ± 74 µmol.min-1.L-1 (37ºC), exceeding those in healthy human serum by 40 and 100 times, respectively. Higher incubation temperature caused a decrease in activity of 20% at 43ºC or 70% at 50ºC for 15 minutes. The ADA lost from 26 to 78% of its activity when hemolymph was pre-incubated at 50ºC for 2 or 15 minutes, respectively. Since the ADA from hemolymph presented high levels, it can be concluded that in healthy and fed animals, adenosine is maintained at low concentrations. In addition, the small variation in activity over the 6.2 to 7.8 range of pH suggests that adenosine is maintained at low levels in hemolymph even under adverse conditions, in which the pH is altered.

Download Full-text

Serum Cholinesterase Variants: Examination of Several Differential Inhibitors, Salts, and Buffers Used to Measure Enzyme Activity

Clinical Chemistry ◽

10.1093/clinchem/17.3.183 ◽

1971 ◽

Vol 17 (3) ◽

pp. 183-191 ◽

Cited By ~ 30

Author(s):

Philip J Garry

Keyword(s):

Enzyme Activity ◽

Sodium Fluoride ◽

Phosphate Buffer ◽

Divalent Cations ◽

Kinetic Studies ◽

Competitive Inhibitor ◽

Serum Cholinesterase ◽

Low Concentrations

Abstract Dibucaine, used as a differential inhibitor with acetyl-, propionyl-, and butyrylthiocholine as substrate, clearly identified the "usual" and "atypical" serum cholinesterases. Succinylcholine was also used successfully as a differential inhibitor with butyrylthiocholine as substrate. Sodium fluoride, used as a differential inhibitor, gave conflicting results, depending on whether Tris or phosphate buffer was used in the assay. Mono- and divalent cations (NaCl, KCl, MgCl2, CaCl2, and BaCl2) activated the "usual" and inhibited the "atypical" enzyme at low concentrations. The "usual" enzyme had the same activity in 0.05 mol of Tris or phosphate buffer per liter, while the heterozygous and "atypical" enzymes showed 12 and 42% inhibition, respectively, when assayed in the phosphate buffer. Kinetic studies showed the phosphate acted as a competitive inhibitor of "atypical" enzyme. Km values, determined for "usual" and "atypical" enzymes, were 0.057 and 0.226 mmol/liter, respectively, with butyrylthiocholine as substrate.

Download Full-text

Porcine glutathione transferase Alpha 2-2 is a human GST A3-3 analogue that catalyses steroid double-bond isomerization

Biochemical Journal ◽

10.1042/bj20100839 ◽

2010 ◽

Vol 431 (1) ◽

pp. 159-167 ◽

Cited By ~ 13

Author(s):

Natalia Fedulova ◽

Françoise Raffalli-Mathieu ◽

Bengt Mannervik

Keyword(s):

Double Bond ◽

Glutathione Transferase ◽

Biological Species ◽

Glutathione Transferases ◽

Special Role ◽

Primary Role ◽

The Novel ◽

Protective Function ◽

Isomerase Activity

A primary role of GSTs (glutathione transferases) is detoxication of electrophilic compounds. In addition to this protective function, hGST (human GST) A3-3, a member of the Alpha class of soluble GSTs, has prominent steroid double-bond isomerase activity. The isomerase reaction is an obligatory step in the biosynthesis of steroid hormones, indicating a special role of hGST A3-3 in steroidogenic tissues. An analogous GST with high steroid isomerase activity has so far not been found in any other biological species. In the present study, we characterized a Sus scrofa (pig) enzyme, pGST A2-2, displaying high steroid isomerase activity. High levels of pGST A2-2 expression were found in ovary, testis and liver. In its functional properties, other than steroid isomerization, pGST A2-2 was most similar to hGST A3-3. The properties of the novel porcine enzyme lend support to the notion that particular GSTs play an important role in steroidogenesis.

Download Full-text

Organ Regeneration on Excised Roots of Chondrilla Juncea and Its Chemical Regulation

Australian Journal of Biological Sciences ◽

10.1071/bi9720691 ◽

1972 ◽

Vol 25 (4) ◽

pp. 691 ◽

Cited By ~ 17

Author(s):

NP Kefford ◽

OH Caso

Keyword(s):

Root Formation ◽

Bud Formation ◽

Endogenous Factors ◽

Chemical Regulation ◽

Synthetic Auxins ◽

Organ Regeneration ◽

Low Concentrations ◽

Standard Assay ◽

Gibberellin Treatment ◽

Regulation Of Regeneration

The effects of endogenous factors (plant age, section length, and section location) and environmental factors (temperature and mineral nutrition) upon organ regeneration on isolated root sections of Ohondrilla juncea L. were used to develop a standard assay system for the study of the chemical regulation of regeneration. Bud and root formation and its polarity in the presence of a variety of regulators alone and in combinations were observed quantitatively. Bud numbers were increased by auxin (low concentrations), cytokinin, and gibberellin treatments. High concentra� tions of auxin inhibited bud formation and this effect was reversed by antiauxin, cytokinin, or gibberellin. Adenine did not counteract auxin� induced bud inhibition but adenine and N�6�benzyladenine did counteract inhibition induced by the purine antagonist 2,6�diaminopurine. Numbers of regenerated roots were increased by auxin treatment and reduced by cytokinin and gibberellin treatment. On control and auxin� treated sections, bud formation was strongly polar and proximal and cytokinin and gibberellin treatments lessened the polarity. Growth retardants inhibited regeneration. Of a number of synthetic auxins tested, 2,4.dichlorophenoxy. acet.O.methylhydroxamic acid and 4�amino.3,5,6.trichloropicolinic acid were the most effective inhibitors of bud formation.

Download Full-text

Plausible molecular mechanism for activation by fumarate and electron transfer of the dopamine β-mono-oxygenase reaction

Biochemical Journal ◽

10.1042/bj20020216 ◽

2002 ◽

Vol 367 (1) ◽

pp. 77-85 ◽

Cited By ~ 4

Author(s):

D. Shyamali WIMALASENA ◽

Samantha P. JAYATILLAKE ◽

Donovan C. HAINES ◽

Kandatege WIMALASENA

Keyword(s):

Electron Transfer ◽

Molecular Mechanism ◽

Geometric Isomer ◽

Enzyme Activation ◽

Reduced Form ◽

Electrostatic Model ◽

Steady State Kinetic ◽

Low Concentrations ◽

Substrate Inhibitor ◽

Inhibition Potency

A series of fumarate analogues has been used to explore the molecular mechanism of the activation of dopamine β-mono-oxygenase by fumarate. Mesaconic acid (MA) and trans-glutaconic acid (TGA) both activate the enzyme at low concentrations, similar to fumarate. However, unlike fumarate, TGA and MA interact effectively with the oxidized enzyme to inhibit it at concentrations of 1—5mM. Monoethylfumarate (EFum) does not activate the enzyme, but inhibits it. In contrast with TGA and MA, however, EFum inhibits the enzyme by interacting with the reduced form. The saturated dicarboxylic acid analogues, the geometric isomer and the diamide of fumaric acid do not either activate or inhibit the enzyme. The phenylethylamine—fumarate conjugate, N-(2-phenylethyl)fumaramide (PEA-Fum), is an 600-fold more potent inhibitor than EFum and behaves as a bi-substrate inhibitor for the reduced enzyme. The amide of PEA-Fum behaves similarly, but with an inhibition potency 20-fold less than that of PEA-Fum. The phenylethylamine conjugates of saturated or geometric isomers of fumarate do not inhibit the enzyme. Based on these findings and on steady-state kinetic analysis, an electrostatic model involving an interaction between the amine group of the enzyme-bound substrate and a carboxylate group of fumarate is proposed to account for enzyme activation by fumarate. Furthermore, in light of the recently proposed model for the similar copper enzyme, peptidylglycine α-hydroxylating mono-oxygenase, the above electrostatic model suggests that fumarate may also play a role in efficient electron transfer between the active-site copper centres of dopamine β-mono-oxygenase.

Download Full-text

Purification and regulatory properties of isocitrate lyase from Escherichia coli ML308

Biochemical Journal ◽

10.1042/bj2500025 ◽

1988 ◽

Vol 250 (1) ◽

pp. 25-31 ◽

Cited By ~ 26

Author(s):

C MacKintosh ◽

H G Nimmo

Keyword(s):

Escherichia Coli ◽

Isocitrate Lyase ◽

Random Order ◽

Kinetic Mechanism ◽

Competitive Inhibitor ◽

Intact Cells ◽

Competing Anions ◽

Effects Of Ph ◽

Order Of Magnitude

Isocitrate lyase was purified to homogeneity from Escherichia coli ML308. Its subunit Mr and native Mr were 44,670 +/- 460 and 17,000-180,000 respectively. The kinetic mechanism of the enzyme was investigated by using product and dead-end inhibitors of the cleavage and condensation reactions. The data indicated a random-order equilibrium mechanism, with formation of a ternary enzyme-isocitrate-succinate complex. In an attempt to predict the properties of isocitrate lyase in intact cells, the effects of pH, inorganic anions and potential regulatory metabolites on the enzyme were studied. The Km of the enzyme for isocitrate was 63 microM at physiological pH and in the absence of competing anions. Chloride, phosphate and sulphate ions inhibited competitively with respect to isocitrate. Phosphoenolpyruvate inhibited non-competitively with respect to isocitrate, but the Ki value suggested that this effect was unlikely to be significant in intact cells. 3-Phosphoglycerate was a competitive inhibitor. At the concentration reported to occur in intact cells, this metabolite would have a significant effect on the activity of isocitrate lyase. The available data suggest that the Km of isocitrate lyase for isocitrate is similar to the concentration of isocitrate in E. coli cells growing on acetate, about one order of magnitude higher than the Km determined in vitro in the absence of competing anions.

Download Full-text

Engineering a Pseudo-26-kDa Schistosoma Glutathione Transferase from bovis/haematobium for Structure, Kinetics, and Ligandin Studies

Biomolecules ◽

10.3390/biom11121844 ◽

2021 ◽

Vol 11 (12) ◽

pp. 1844

Author(s):

Neo Padi ◽

Blessing Oluebube Akumadu ◽

Olga Faerch ◽

Chinyere Aloke ◽

Vanessa Meyer ◽

...

Keyword(s):

Glutathione Transferase ◽

Specific Activity ◽

Enzyme Stability ◽

Competitive Inhibitor ◽

Spectroscopic Studies ◽

Complete Sequence ◽

Detoxification Enzymes ◽

Ligand Docking ◽

Sequence Information ◽

Dimer Interface

Glutathione transferases (GSTs) are the main detoxification enzymes in schistosomes. These parasitic enzymes tend to be upregulated during drug treatment, with Schistosoma haematobium being one of the species that mainly affect humans. There is a lack of complete sequence information on the closely related bovis and haematobium 26-kDa GST isoforms in any database. Consequently, we engineered a pseudo-26-kDa S. bovis/haematobium GST (Sbh26GST) to understand structure–function relations and ligandin activity towards selected potential ligands. Sbh26GST was overexpressed in Escherichia coli as an MBP-fusion protein, purified to homogeneity and catalyzed 1-chloro-2,4-dinitrobenzene-glutathione (CDNB-GSH) conjugation activity, with a specific activity of 13 μmol/min/mg. This activity decreased by ~95% in the presence of bromosulfophthalein (BSP), which showed an IC50 of 27 µM. Additionally, enzyme kinetics revealed that BSP acts as a non-competitive inhibitor relative to GSH. Spectroscopic studies affirmed that Sbh26GST adopts the canonical GST structure, which is predominantly α-helical. Further extrinsic 8-anilino-1-naphthalenesulfonate (ANS) spectroscopy illustrated that BSP, praziquantel (PZQ), and artemisinin (ART) might preferentially bind at the dimer interface or in proximity to the hydrophobic substrate-binding site of the enzyme. The Sbh26GST-BSP interaction is both enthalpically and entropically driven, with a stoichiometry of one BSP molecule per Sbh26GST dimer. Enzyme stability appeared enhanced in the presence of BSP and GSH. Induced fit ligand docking affirmed the spectroscopic, thermodynamic, and molecular modelling results. In conclusion, BSP is a potent inhibitor of Sbh26GST and could potentially be rationalized as a treatment for schistosomiasis.

Download Full-text