scholarly journals Participation of the phenolic hydroxyl group of Tyr-8 in the catalytic mechanism of human glutathione transferase P1-1

1992 ◽  
Vol 285 (2) ◽  
pp. 537-540 ◽  
Author(s):  
R H Kolm ◽  
G E Sroga ◽  
B Mannervik
Keyword(s):  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Specific Activity ◽  
Dissociation Constants ◽  
Phenolic Hydroxyl ◽  
Mutant Enzyme ◽  
Hydroxyl Group ◽  
Coding Region ◽  
Ph Profile ◽  
Ionic Dissociation

The coding region of cDNA corresponding to human class Pi glutathione transferase P1-1 was amplified by the PCR, subcloned into an expression vector, pKHP1, expressed in Escherichia coli, and characterized. The physicochemical and catalytic properties of recombinant glutathione transferase P1-1 were indistinguishable from those of the enzyme previously isolated from human placenta. The active-site residue Tyr-8 of the wild-type enzyme was converted into Phe by means of oligonucleotide-directed mutagenesis. The mutant enzyme Y8F displayed a 300-fold decrease in specific activity, ascribable mainly to a lowered k(cat.) (or V) value. Kinetic parameters reflecting binding affinity, S0.5 (substrate concn. giving 1/2V) and I50 (concn. of inhibitor giving 50% remaining activity), were only moderately elevated in the mutant enzyme. These results indicate that Tyr-8 contributes primarily to catalysis as such, rather than to binding of the substrates. The dependence of k(cat.)/Km on pH shows an optimum at pH 7.0, defined by acidic and basic ionic dissociation constants with pKa1 = 6.7 and pKa2 = 7.3 respectively. The mutant enzyme Y8F does not display the basic limb of the k(cat.)/Km versus pH profile, but shows a monotonic increase of k(cat.)/Km with an apparent pKa1 of 7.1. The results indicate that the phenolic hydroxyl group of Tyr-8 in un-ionized form, but not the phenolate of Tyr-8, contributes to catalysis by glutathione transferase P1-1.

scholarly journals The Role of Glutathione in the Isomerization of Δ5-Androstene- 3,17-dione Catalyzed by Human Glutathione Transferase A1-1

2001 ◽  
Vol 276 (15) ◽  
pp. 11698-11704 ◽  
Author(s):  
Pär L. Pettersson ◽  
Bengt Mannervik
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Ph Dependence ◽  
Enzymatic Catalysis ◽  
Catalytic Efficiency ◽  
Protonated Form ◽  
Hydroxyl Group ◽  
Isomerization Reaction

Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Δ5-androstene-3,17-dione (AD) into Δ4-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited catalytic effect.S-Alkylglutathione derivatives do not promote the reaction, and the pH dependence of the isomerization indicates that the glutathione thiolate serves as a base in the catalytic mechanism. Mutation of the active-site Tyr9into Phe significantly decreases the steady-state kinetic parameters, alters their pH dependence, and increases the pKavalue of the enzyme-bound glutathione thiol. Thus, Tyr9promotes the reaction via its phenolic hydroxyl group in protonated form. GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than GST A2-2 and GST A4-4, both having a glutathione cofactor and an active-site Tyr9residue. The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction. The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3β-hydroxysteroid dehydrogenase.

scholarly journals Contribution of five amino acid residues in the glutathione-binding site to the function of human glutathione transferase P1-1

1992 ◽  
Vol 285 (2) ◽  
pp. 377-381 ◽  
Author(s):  
M Widersten ◽  
R H Kolm ◽  
R Björnestedt ◽  
B Mannervik
Keyword(s):  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Catalytic Efficiency ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Ph Profile ◽  
Mutant Proteins ◽  
Catalytically Active ◽  
Active Site Cavity ◽  
Important Structure

Five amino acids in proximity to GSH bound in the active-site cavity of human Class Pi glutathione transferase (GST) P1-1 were mutated by oligonucleotide-directed site-specific mutagenesis. The following mutations gave catalytically active mutant proteins with the proper dimeric structure: Arg14----Ala, Lys45----Ala, Gln52----Ala, Gln65----His and Asp99----Asn. The mutation Gln65----Ala was also made, but the protein was not characterized because of its poor catalytic activity. Residues Arg14, Lys45, Gln52 and Gln65 all contribute to binding of glutathione, and the substitutions caused an approx. 10-fold decrease in affinity, corresponding to 5 kJ/mol, except for Arg14, for which the effect was larger. In addition, Arg14 appears to have an important structure role, since the Arg14----Ala mutant demonstrated a significantly lower stability as compared with the wild-type and the other mutant enzymes. Asp99 primarily contributes to catalysis rather than to binding. The kcat./Km-versus-pH profile for the Asp99----Asn mutant is shifted by 0.5 pH unit in the alkaline direction, and it is proposed that Asp99 may participate in proton transfer in the catalytic mechanism. The possibility of redesigning the substrate specificity for GSTs was shown by the fact that the mutant Lys45----Ala displayed a higher catalytic efficiency with GSH monoethyl ester than with its natural substrate, GSH.

Multifunctional Role of Tyr 108 in the Catalytic Mechanism of Human Glutathione Transferase P1-1. Crystallographic and Kinetic Studies on the Y108F Mutant Enzyme†,‡

Biochemistry ◽  
1997 ◽  
Vol 36 (20) ◽  
pp. 6207-6217 ◽  
Author(s):  
Mario Lo Bello ◽  
Aaron J. Oakley ◽  
Andrea Battistoni ◽  
Anna P. Mazzetti ◽  
Marzia Nuccetelli ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Kinetic Studies ◽  
Mutant Enzyme

scholarly journals Mutagenesis of the active site of the human Theta-class glutathione transferase GSTT2-2: catalysis with different substrates involves different residues

1996 ◽  
Vol 319 (1) ◽  
pp. 315-321 ◽  
Author(s):  
Kian-Leong TAN ◽  
Gareth CHELVANAYAGAM ◽  
Michael W. PARKER ◽  
Philip G. BOARD
Keyword(s):  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Specific Activity ◽  
Cumene Hydroperoxide ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Serine Residue ◽  
Subsequent Removal ◽  
Carbonium Ion

The role of serine-11 in the catalytic mechanism of recombinant human GSTT2-2 was examined by site-directed mutagenesis. Amino acid sequence comparison of the Theta-class isoenzymes has identified a conserved serine residue in the N-terminal domain [Wilce, Board, Feil and Parker (1995) EMBO J. 14, 2133–2143]. This conserved serine has been implicated in the activation of the enzyme-bound glutathione [Board, Coggan and Parker (1995) Biochem. J. 311, 247–250]. Mutating the equivalent serine (residue 11) of GSTT2-2 to Ala, Thr or Tyr abolished the catalytic properties of GSTT2-2 with cumene hydroperoxide and ethacrynic acid as second substrate. However, with 1-menaphthyl sulphate (MSu) as the second substrate, the specific activity of the S11A mutant was doubled, while the S11T mutant retained half the wild-type activity and the S11Y mutant was inactive. The role of Ser-11 in catalysis seems to vary with different second substrates. In the substitution reaction with MSu, GSTT2-2 activity appears to depend on the size of the Ser-11 replacement rather than the presence of a side-chain hydroxy group. In addition, the reaction rate appears to be a function of pH, and there is no non-enzymic reaction even at high pH. We demonstrated that a reaction between MSu and an alternative thiol such as L-cysteine or 2-mercaptoethanol can take place in the presence of S-methylglutathione and GSTT2-2. We propose that the catalytic activity of GSTT2-2 with MSu is preceded by a conformational or charge modification to the enzyme upon the binding of glutathione or S-methylglutathione. This is followed by the binding of MSu and the subsequent removal of the sulphate group, giving rise to the carbonium ion of 1-methylnaphthelene as the electrophile that reacts with the nucleophilic species. The reaction mechanism of GSTT2-2 with MSu may represent a novel function of GSTT2-2 as a glutathione-dependent sulphatase.

The Influence of Anthraquinone in Sulfite Delignification of Norway Spruce. 2. Phenolic Hydroxyl Group Formation

Holzforschung ◽  
1994 ◽  
Vol 48 (s1) ◽  
pp. 140-145 ◽  
Author(s):  
Hsin-Tai Chen ◽  
Masamitsu Funaoka ◽  
Yuan-Zong Lai
Keyword(s):  
Norway Spruce ◽  
Group Formation ◽  
Phenolic Hydroxyl ◽  
Hydroxyl Group

scholarly journals ACID-BASE PROPERTIES OF -UNSATURATED KETONES OBTAINED FROM REACTION OF 5-FORMYL-8-HYDROXYQUINOLINE WITH P-SUBSTITUTED ACETOPHENONES

2019 ◽  
Vol 16 (31) ◽  
pp. 755-764
Author(s):  
Roberto FERNANDEZ-MAESTRE ◽  
Alonso J MARRUGO-GONZÁLEZ
Keyword(s):  
Electron Donor ◽  
Opposite Effect ◽  
Dissociation Constants ◽  
Phenyl Group ◽  
Hydroxyl Group ◽  
Ultraviolet Spectroscopy ◽  
Acid Base ◽  
Unsaturated Ketones ◽  
Ph Values ◽  
Phenyl Fragment

Chalcones (α,β-unsaturated ketones) containing aromatic or heterocyclic radicals are highly reactive, allowing the synthesis of novel organic compounds. In this study, the dissociation constants (pKa) of seven chalcones derived from 8-hydroxyquinoline were determined and the influence on dissociation of substituents in the phenyl group (-CH3, -OCH3, -N(CH3)2, -Cl, -Br, and -NO2) was analysed. pKa values are important because they determine the pH at which ligands are fully deprotonated -when they show their maximum chelating properties- and determine the ligands interactions at different pH values. The chalcones’ pKa’s were calculated by visible ultraviolet spectroscopy in a water-ethanol (1:1) mixture using the Henderson-Hasselbach equation. It was shown that the 8-hydroxyquinolinic fragment has a large electron donor effect on the π system of the chalcones. The introduction of substituents (R) in the phenyl fragment of the chalcones slightly affected the dissociation of the hydroxyl group and the protonation of the nitrogen in the hydroxyquinoline fragment. The acceptor substituents (Cl, Br, NO2) increased the polarity of OH- and its acidity. Nitrogen protonation decreased electron donor properties of this fragment, and deprotonation of the hydroxyl caused the opposite effect. Substituents introduction in the phenyl fragment slightly affected hydroxyl group dissociation and nitrogen protonation.

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

Biomolecules ◽  
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.

scholarly journals pH modelling in aquatic systems with time-variable acid-base dissociation constants applied to the turbid, tidal Scheldt estuary

2009 ◽  
Vol 6 (8) ◽  
pp. 1539-1561 ◽  
Author(s):  
A. F. Hofmann ◽  
J. J. Middelburg ◽  
K. Soetaert ◽  
F. J. R. Meysman
Keyword(s):  
Dissolved Inorganic Carbon ◽  
Dissociation Constants ◽  
Time Variable ◽  
Scheldt Estuary ◽  
Biogeochemical Processes ◽  
Acid Base ◽  
Ph Profile ◽  
Proton Production ◽  
Co2 Degassing

Abstract. A new pH modelling approach is presented that explicitly quantifies the influence of biogeochemical processes on proton cycling and pH in an aquatic ecosystem, and which accounts for time variable acid-base dissociation constants. As a case study, the method is applied to investigate proton cycling and long-term pH trends in the Scheldt estuary (SW Netherlands, N Belgium). This analysis identifies the dominant biogeochemical processes involved in proton cycling in this heterotrophic, turbid estuary. Furthermore, information on the factors controlling the longitudinal pH profile along the estuary as well as long-term pH changes are obtained. Proton production by nitrification is identified as the principal biological process governing the pH. Its acidifying effect is mainly counteracted by proton consumption due to CO2 degassing. Overall, CO2 degassing generates the largest proton turnover in the whole estuary on a yearly basis. The main driver of long-term changes in the mean estuarine pH over the period 2001 to 2004 is the decreasing freshwater flow, which influences the pH directly via a decreasing supply of dissolved inorganic carbon and alkalinity, and also indirectly, via decreasing ammonia loadings and lower nitrification rates.

Aqueous Medium Radical Addition to Ketimines with a Phenolic Hydroxyl Group

Synlett ◽  
2004 ◽  
Vol 2004 (14) ◽  
pp. 2597-2599 ◽  
Author(s):  
Yoshiji Takemoto ◽  
Hideto Miyabe ◽  
Yousuke Yamaoka
Keyword(s):  
Aqueous Medium ◽  
Radical Addition ◽  
Phenolic Hydroxyl ◽  
Hydroxyl Group
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