The catalytic mechanism of glutathione S-transferase (GST). Spectroscopic determination of the pKa of Tyr-9 in rat alpha 1-1 GST.

Atkins, William M.; Wang, R. W.; Bird, A. W.; Newton, Deborah J.; Lu, Anthony Y. H.

doi:10.1016/s0021-9258(19)36496-8

Cited by 93 publications

(38 citation statements)

References 24 publications

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“…The fluorescence emission of tyrosine side chains is remarkably sensitive to the hydrogen-bonding environment, and it has been established that the extent of charge-transfer in a hydrogen-bonded complex is dependent on the basicity of the acceptor and on the solvent polarity (Ratajczak, 1972;Willis & Szabo, 1991). Here we extend our previous studies (Atkins et al, 1993) with the engineered rat alpha 1-1 GSTs, W21F and W21F:Y9F, which contain no tryptophans. Together these mutants allow for characterization of the local environment of Tyr-9 in the presence of various ligands.…”

supporting

confidence: 62%

“…Direct comparison of the correlated excitation-emission spectra of the ligand-free W21F and the W21F:Y9F mutant proteins indicates that, at pH 7.5, the single mutant has a strong emission "cross-peak" centered at 335-340 nm, coupled to an excitation band at ∼255 nm and a much less intense excitation band at ∼300 nm, which appears as a "lobe" on the long-wavelength side of the peak at 280 nm along the excitation axis; both of these cross-peaks are due apparently to a contribution from Tyr-9, because they are absent in the correlated contours of the W21F:Y9F protein (Figure 1). Because the emission maximum of tyrosine that is hydrogen bonded to "bulk" water (305-310 nm) is insensitive to changes in the dielectric constant of the environment [Willis and Szabo (1991) and data not shown], we propose that the cross-peaks in the correlated spectrum of the W21F mutant arise because Tyr-9 is partially deprotonated at pH 7.5 (Atkins et al, 1993), in the absence of GSH. This is based on the well-established absorbance and emission properties of tyrosine.…”

Section: Resultsmentioning

confidence: 89%

“…Substrate-Free and GSH-Bound Glutathione S-Transferase. We have described previously the mutant rat A1-1 GST proteins W21F and W21F:Y9F, in which the single tryptophan found in the wild protein has been replaced with the fluorimetrically silent phenylalanine (Atkins et al, 1993). In addition, the W21F:Y9F mutant has a phenylalanine substituted for the active site Tyr-9.…”

Section: Resultsmentioning

confidence: 99%

“…Replacement of Trp-21 with phenylalanine has negligible effect on the catalytic properties of the enzyme (Wang et al, 1992b). Together, these proteins allow for the characterization of the active site tyrosine (Atkins et al, 1993). The previous characterization was specifically aimed at determining the pK a of Tyr-9.…”

Section: Resultsmentioning

confidence: 99%

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Ligand Effects on the Fluorescence Properties of Tyrosine-9 in Alpha 1-1 Glutathione S-Transferase

Dietze

Wang

et al. 1996

Biochemistry

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A conserved tyrosine plays a critical role in catalysis by mammalian glutathione S-transferases (GSTs) of the alpha-, mu-, and pi-classes, by forming a hydrogen bond to and stabilizing the thiolate form of glutathione. The hydrogen bonding properties of this tyrosine in the rat A1-1 GST (Tyr-9), in the absence and presence of ligands, have been studied by steady state and time-resolved fluorescence spectroscopy. In order to achieve this, the single tryptophan (Trp 21) found in the rat A1-1 GST has been replaced with the fluorometrically silent phenylalanine (W21F). Additionally, a double mutant lacking this tryptophan and the catalytic tyrosine (W21F:Y9F) has been constructed, and these mutants have been used as probes of ligand effects at Tyr-9. A comparison of the correlated excitation--emission spectra of the W21F mutant and the W21F-Y9F indicates that a red-shifted emission component is contributed by Tyr-9 with excitation bands at 255 and 300 nm, in the ligand-free enzyme. The pH-dependence of the intensity of these spectral cross-peaks is consistent with an active site tyrosine with a pKa of 8.1-8.3. Upon addition of GSH, the red-shifted component is quenched. Multifrequency phase/modulation fluorescence experiments qualitatively demonstrate that GSH causes a decrease in the average excited state lifetime on the red-edge of the spectrum of W21F but not of the W21F:Y9F spectrum. Steady state correlated difference spectra (W21F-W21F:Y9F) have been used to obtain a model for the excitation-emission correlated spectrum of Tyr-9, which indicates that Tyr-9 is heterogeneous at pH 7.5, with properties of both tyrosinate and "normal tyrosine". The tyrosinate fraction is eliminated, and the blue-shifted component becomes more intense upon addition of GSH conjugates, indicating that the weak hydrogen bond between Tyr-9 and thioethers has little charge-transfer character. The S-methyl GSH yields an "anomalous" spectrum at pH 7.5, which retains cross-peaks consistent with ionized tyrosinate. These results indicate that, in the absence of ligand, Tyr-9 forms a strongly polarized hydrogen bond or a fraction of the phenolic hydroxyl group is partially deprotonated. However, when a GSH conjugate with a sufficiently large hydrophobic group occupies the H-site, Tyr-9 is fully protonated, with little charge-transfer character.

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supporting

confidence: 62%

Section: Resultsmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Ligand Effects on the Fluorescence Properties of Tyrosine-9 in Alpha 1-1 Glutathione S-Transferase

Dietze

Wang

et al. 1996

Biochemistry

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“…This residue is presumably the main structural determinant conferring tight binding. A similar profile has been observed by studying the pH dependence of the kinetic parameters of alpha-class GSTs [34,35].…”

Section: The Effect Of Ph Temperature and Viscosity On Ic50supporting

confidence: 76%

The Interaction of the Flavonoid Fisetin with Human Glutathione Transferase A1-1

et al. 2021

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Glutathione transferases (GSTs) are a family of Phase II detoxification enzymes that are involved in the development of the multidrug resistance (MDR) mechanism in cancer cells and therefore affect the clinical outcome of cancer chemotherapy. The discovery of nontoxic natural compounds as inhibitors for GSTs is a promising approach for chemosensitizing and reversing MDR. Fisetin (7,3′,4′-flavon-3-ol) is a plant flavonol present in many plants and fruits. In the present work, the interaction of fisetin with human glutathione transferase A1-1 (hGSTA1-1) was investigated. Kinetic analysis revealed that fisetin is a reversible inhibitor for hGSTA1-1 with IC50 1.2 ± 0.1 μΜ. It functions as a mixed-type inhibitor toward glutathione (GSH) and as a noncompetitive inhibitor toward the electrophile substrate 1-chloro-2,4-dinitrobenzene (CDNB). In silico molecular modeling and docking predicted that fisetin binds at a distinct location, in the solvent channel of the enzyme, and occupies the entrance of the substrate-binding sites. Treatment of proliferating human epithelial colorectal adenocarcinoma cells (CaCo-2) with fisetin causes a reduction in the expression of hGSTA1-1 at the mRNA and protein levels. In addition, fisetin inhibits GST activity in CaCo-2 cell crude extract with an IC50 (2.5 ± 0.1 μΜ), comparable to that measured using purified recombinant hGSTA1-1. These actions of fisetin can provide a synergistic role toward the suppression and chemosensitization of cancer cells. The results of the present study provide insights into the development of safe and effective GST-targeted cancer chemosensitizers.

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On the reaction mechanism of class Pi glutathione S-transferase

Orozco¹,

Vega²,

Parraga³

et al. 1997

Proteins

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Theoretical calculations were performed to examine the ionization of the phenolic group of Tyr7 and the thiol group of glutathione in aqueous solution and in the protein class-pi glutathione S-transferase (GST-Pi). Three model systems were considered for simulations in the protein environments the free enzyme, the complex between glutathione and the enzyme, and the complex between 1-chloro-2.4-dinitrobenzene, glutathione, and the enzyme. The structures derived from Molecular Dynamics simulations were compared with the crystallographic data available for the complex between the inhibitor S-(p-nitrobenzyl)glutathione and GST-Pi, the glutathione-bound form of GST-Pi, and the free enzyme carboxymethylated in Cys47. Free-energy perturbation techniques were used to determine the thermodynamics quantities for ionization of the phenol and thiol groups. The functional implications of Tyr7 in the activation of the glutathione thiol group are discussed in the light of present results, which in agreement with previous studies suggest that Tyr7 in un-ionized form contributes to the catalytic process of glutathione S-transferase, the thiolate anion being stabilized by hydrogen bond with Tyr7 and by interactions with hydrating water molecules.

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The catalytic mechanism of glutathione S-transferase (GST). Spectroscopic determination of the pKa of Tyr-9 in rat alpha 1-1 GST.

Cited by 93 publications

References 24 publications

Ligand Effects on the Fluorescence Properties of Tyrosine-9 in Alpha 1-1 Glutathione S-Transferase

Ligand Effects on the Fluorescence Properties of Tyrosine-9 in Alpha 1-1 Glutathione S-Transferase

The Interaction of the Flavonoid Fisetin with Human Glutathione Transferase A1-1

On the reaction mechanism of class Pi glutathione S-transferase

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