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

Role of Lys-226 in the Catalytic Mechanism ofBacillus StearothermophilusSerine HydroxymethyltransferaseCrystal Structure and Kinetic Studies†,‡

Biochemistry ◽  
2005 ◽  
Vol 44 (18) ◽  
pp. 6929-6937 ◽  
Author(s):  
Siddegowda Bhavani ◽  
V. Trivedi ◽  
V. R. Jala ◽  
H. S. Subramanya ◽  
Purnima Kaul ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Kinetic Studies

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.

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.

Role of the Glutamyl α-Carboxylate of the Substrate Glutathione in the Catalytic Mechanism of Human Glutathione Transferase A1-1†

Biochemistry ◽  
2001 ◽  
Vol 40 (51) ◽  
pp. 15835-15845 ◽  
Author(s):  
Ann Gustafsson ◽  
Pär L. Pettersson ◽  
Leif Grehn ◽  
Per Jemth ◽  
Bengt Mannervik
Keyword(s):  
Catalytic Mechanism ◽  
Glutathione Transferase

Crystal structures and kinetic studies of human Kappa class glutathione transferase provide insights into the catalytic mechanism

2011 ◽  
Vol 439 (2) ◽  
pp. 215-225 ◽  
Author(s):  
Bing Wang ◽  
Yingjie Peng ◽  
Tianlong Zhang ◽  
Jianping Ding
Keyword(s):  
Binding Site ◽  
Crystal Structures ◽  
Cellular Localization ◽  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Substrate Binding ◽  
Kinetic Studies ◽  
Thiol Group ◽  
Kinetic Mechanism ◽  
Cellular Detoxification

GSTs (glutathione transferases) are a family of enzymes that primarily catalyse nucleophilic addition of the thiol of GSH (reduced glutathione) to a variety of hydrophobic electrophiles in the cellular detoxification of cytotoxic and genotoxic compounds. GSTks (Kappa class GSTs) are a distinct class because of their unique cellular localization, function and structure. In the present paper we report the crystal structures of hGSTk (human GSTk) in apo-form and in complex with GTX (S-hexylglutathione) and steady-state kinetic studies, revealing insights into the catalytic mechanism of hGSTk and other GSTks. Substrate binding induces a conformational change of the active site from an ‘open’ conformation in the apo-form to a ‘closed’ conformation in the GTX-bound complex, facilitating formations of the G site (GSH-binding site) and the H site (hydrophobic substrate-binding site). The conserved Ser16 at the G site functions as the catalytic residue in the deprotonation of the thiol group and the conserved Asp69, Ser200, Asp201 and Arg202 form a network of interactions with γ-glutamyl carboxylate to stabilize the thiolate anion. The H site is a large hydrophobic pocket with conformational flexibility to allow the binding of different hydrophobic substrates. The kinetic mechanism of hGSTk conforms to a rapid equilibrium random sequential Bi Bi model.

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 soluble glutathione transferase superfamily: role of Mu class in triclabendazole sulphoxide challenge in Fasciola hepatica

2021 ◽  
Vol 120 (3) ◽  
pp. 979-991
Author(s):  
Rebekah B. Stuart ◽  
Suzanne Zwaanswijk ◽  
Neil D. MacKintosh ◽  
Boontarikaan Witikornkul ◽  
Peter M. Brophy ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Liver Fluke ◽  
Glutathione Transferase ◽  
Fasciola Hepatica ◽  
Affinity Purification ◽  
Economic Loss ◽  
Differential Abundance ◽  
Parasitic Helminths

AbstractFasciola hepatica (liver fluke), a significant threat to food security, causes global economic loss for the livestock industry and is re-emerging as a foodborne disease of humans. In the absence of vaccines, treatment control is by anthelmintics; with only triclabendazole (TCBZ) currently effective against all stages of F. hepatica in livestock and humans. There is widespread resistance to TCBZ and its detoxification by flukes might contribute to the mechanism. However, there is limited phase I capacity in adult parasitic helminths with the phase II detoxification system dominated by the soluble glutathione transferase (GST) superfamily. Previous proteomic studies have demonstrated that the levels of Mu class GST from pooled F. hepatica parasites respond under TCBZ-sulphoxide (TCBZ-SO) challenge during in vitro culture ex-host. We have extended this finding by exploiting a sub-proteomic lead strategy to measure the change in the total soluble GST profile (GST-ome) of individual TCBZ-susceptible F. hepatica on TCBZ-SO-exposure in vitro culture. TCBZ-SO exposure demonstrated differential abundance of FhGST-Mu29 and FhGST-Mu26 following affinity purification using both GSH and S-hexyl GSH affinity. Furthermore, a low or weak affinity matrix interacting Mu class GST (FhGST-Mu5) has been identified and recombinantly expressed and represents a new low-affinity Mu class GST. Low-affinity GST isoforms within the GST-ome was not restricted to FhGST-Mu5 with a second likely low-affinity sigma class GST (FhGST-S2) uncovered. This study represents the most complete Fasciola GST-ome generated to date and has supported the potential of subproteomic analyses on individual adult flukes.

Study of the catalytic mechanism of a non-heme Fe catalyst: The role of the spin state of the iron

2021 ◽  
Vol 764 ◽  
pp. 138282
Author(s):  
Aikaterini Gemenetzi ◽  
Panagiota Stathi ◽  
Yiannis Deligiannakis ◽  
Maria Louloudi
Keyword(s):  
Spin State ◽  
Catalytic Mechanism ◽  
Fe Catalyst
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