A Highly Acidic Tyrosine 9 and a Normally Titrating Tyrosine 212 Contribute to the Catalytic Mechanism of Human Glutathione Transferase A4-4

2001 ◽  
Vol 280 (3) ◽  
pp. 878-882 ◽  
Author(s):  
Ina Hubatsch ◽  
Bengt Mannervik
Keyword(s):  
Catalytic Mechanism ◽  
Glutathione Transferase

Design of a graphene oxide-BODIPY conjugate for glutathione depletion and photodynamic therapy

2D Materials ◽  
2021 ◽  
Author(s):  
Giacomo Reina ◽  
Amalia Ruiz ◽  
Barbara Richichi ◽  
Giacomo Biagiotti ◽  
Gina Elena Giacomoazzo ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Photodynamic Therapy ◽  
Cell Viability ◽  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Bathochromic Shift ◽  
3D Models ◽  
Glutathione Depletion ◽  
In Vitro Biocompatibility

Abstract Boron dipyrromethene derivates (BODIPYs) are promising photosensitisers (PSs) for cancer treatment using photodynamic therapy (PDT). This study investigates the functionalisation of graphene oxide (GO) with a BODIPY derivate for glutathione (GSH) depletion and PDT. The functionalisation of GO with a 3,5-dichloro-8-(4-boronophenyl) BODIPY via a diol derivatisation with the phenyl boronic acid moiety at the meso position of the BODIPY core, allowed to preserve the intrinsic properties of GO. We demonstrated that both chlorine atoms were substituted by GSH in the presence of glutathione transferase (GST), inducing a relevant bathochromic shift in the absorption/emission features and thus generating the active PS. Ex vitro assessment using cell lysates containing cytoplasmatic GST revealed the intracellular catalytic mechanism for the nucleophilic substitution of the GO-BODIPY adduct with GSH. Confocal microscopy studies showed important differences in the cellular uptake of free BODIPY and GO-BODIPY and revealed the coexistence of GO-BODIPY, GO-BODIPY-GS, and GO-BODIPY-GS2 species inside vesicles and in the cytoplasm of the cells after 24 h of incubation. In vitro biocompatibility and safety of GO and GO-BODIPY were evaluated in 2D and 3D models of prostate adenocarcinoma cells (PC-3), where no toxicity was observed up to 100 µg/mL of GO/GO-BODIPY in all treated groups 24 h post-treatment (cell viability > 90%). Only a slight decrease to 80% at 100 µg/mL was observed after 48 h of incubation. We demonstrated the efficacy of a GO adduct containing an α-chlorine-substituted BODIPY for the simultaneous depletion of intracellular GSH and the photogeneration of reactive oxygen species using a halogen white light source (5.4 mW/cm2) with a maximum in the range of 500-800 nm, which significantly reduced cell viability (< 50%) after irradiation. Our study provides a new vision on how to apply BODIPY derivates and potentiate the toxicity of PDT in prostate and other types of cancer.

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.

scholarly journals Mu-class glutathione transferase from Xenopus laevis: molecular cloning, expression and site-directed mutagenesis

2002 ◽  
Vol 365 (3) ◽  
pp. 685-691 ◽  
Author(s):  
Antonella De LUCA ◽  
Bartolo FAVALORO ◽  
Stefania ANGELUCCI ◽  
Paolo SACCHETTA ◽  
Carmine Di ILIO
Keyword(s):  
Xenopus Laevis ◽  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Structural Instability ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Calculated Molecular Mass

A cDNA encoding a Mu-class glutathione transferase (XlGSTM1-1) has been isolated from a Xenopus laevis liver library, and its nucleotide sequence has been determined. XlGSTM1-1 is composed of 219 amino acid residues with a calculated molecular mass of 25359Da. Unlike many mammalian Mu-class GSTs, XlGSTM1-1 has a narrow spectrum of substrate specificity and it is also less effective in conjugating 1-chloro-2,4-dinitrobenzene. A notable structural feature of XlGSTM1-1 is the presence of the Cys-139 residue in place of the Glu-139, as well as the absence of the Cys-114 residue, present in other Mu-class GSTs, which is replaced by Ala. Site-directed mutagenesis experiments indicate that Cys-139 is not involved in the catalytic mechanism of XlGSTM1-1 but may be in part responsible for its structural instability, and experiments in vivo confirmed the role of this residue in stability. Evidence indicating that Arg-107 is essential for the 1-chloro-2,4-dinitrobenzene conjugation capacity of XlGSTM1-1 is also presented.

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 Structural and Functional Characterization of Camelus dromedarius Glutathione Transferase M1-1

Life ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 106
Author(s):  
Fereniki Perperopoulou ◽  
Nirmal Poudel ◽  
Anastassios C. Papageorgiou ◽  
Farid S. Ataya ◽  
Nikolaos E. Labrou
Keyword(s):  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Functional Characterization ◽  
Large Family ◽  
Camelus Dromedarius ◽  
X Ray Crystallography ◽  
Differential Scanning Fluorimetry ◽  
Multifunctional Enzymes ◽  
Xenobiotic Compounds

Glutathione transferases (GSTs; EC. 2.5.1.18) are a large family of multifunctional enzymes that play crucial roles in the metabolism and inactivation of a broad range of xenobiotic compounds. In the present work, we report the kinetic and structural characterization of the isoenzyme GSTM1-1 from Camelus dromedarius (CdGSTM1-1). The CdGSΤM1-1 was expressed in E. coli BL21 (DE3) and was purified by affinity chromatography. Kinetics analysis showed that the enzyme displays a relative narrow substrate specificity and restricted ability to bind xenobiotic compounds. The crystal structures of CdGSΤM1-1 were determined by X-ray crystallography in complex with the substrate (GSH) or the reaction product (S-p-nitrobenzyl-GSH), providing snapshots of the induced-fit catalytic mechanism. The thermodynamic stability of CdGSTM1-1 was investigated using differential scanning fluorimetry (DSF) in the absence and in presence of GSH and S-p-nitrobenzyl-GSH and revealed that the enzyme’s structure is significantly stabilized by its ligands. The results of the present study advance the understanding of camelid GST detoxification mechanisms and their contribution to abiotic stress adaptation in harsh desert conditions.

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