Rapid kinetic, spectroscopic, and potentiometric studies have been performed on human Theta class glutathione transferase T2-2 to dissect the mechanism of interaction of this enzyme with its natural substrate GSH. Theta class glutathione transferases are considered to be older than Alpha, Pi, and Mu classes in the evolutionary pathway. As in the more recently evolved GSTs, the activation of GSH in the human Theta enzyme proceeds by a forced deprotonation of the sulfhydryl group (pK(alpha) = 6.1). The thiol proton is released quantitatively in solution, but above pH 6.5, a protein residue acts as an internal base. Unlike Alpha, Mu, and Pi class isoenzymes, the GSH-binding mechanism occurs via a simple bimolecular reaction with k(on) and k(off) values at least hundred times lower (k(on) = (2.7 +/- 0.8) x 10(4) M-1 s(-1) k(off) = 36 +/- 9 s(-1), at 37 degreesC). Replacement of Arg-107 by alanine, using site-directed mutagenesis, remarkably increases the pK alpha value of the bound GSH and modifies the substrate binding modality. Y107A mutant enzyme displays a mechanism and rate constants for GSH binding approaching those of Alpha, Mu, and Pi isoenzymes. Comparison of available crystallographic data for all these GSTs reveals an unexpected evolutionary trend in terms of flexibility, which provides a basis for understanding our experimental results.

Caccuri, A.M., Antonini, G., Board, P.G., Flanagan, J., Parker, M.W., Paolesse, R., et al. (2001). Human glutathione transferase T2-2 discloses some evolutionary strategies for optimization of substrate binding to the active site of glutathione transferases. THE JOURNAL OF BIOLOGICAL CHEMISTRY, 276(8), 5427-5431 [10.1074/jbc.M002819200].

Human glutathione transferase T2-2 discloses some evolutionary strategies for optimization of substrate binding to the active site of glutathione transferases

CACCURI, ANNA MARIA;PAOLESSE, ROBERTO;FEDERICI, GIORGIO;LO BELLO, MARIO;RICCI, GIORGIO
2001

Abstract

Rapid kinetic, spectroscopic, and potentiometric studies have been performed on human Theta class glutathione transferase T2-2 to dissect the mechanism of interaction of this enzyme with its natural substrate GSH. Theta class glutathione transferases are considered to be older than Alpha, Pi, and Mu classes in the evolutionary pathway. As in the more recently evolved GSTs, the activation of GSH in the human Theta enzyme proceeds by a forced deprotonation of the sulfhydryl group (pK(alpha) = 6.1). The thiol proton is released quantitatively in solution, but above pH 6.5, a protein residue acts as an internal base. Unlike Alpha, Mu, and Pi class isoenzymes, the GSH-binding mechanism occurs via a simple bimolecular reaction with k(on) and k(off) values at least hundred times lower (k(on) = (2.7 +/- 0.8) x 10(4) M-1 s(-1) k(off) = 36 +/- 9 s(-1), at 37 degreesC). Replacement of Arg-107 by alanine, using site-directed mutagenesis, remarkably increases the pK alpha value of the bound GSH and modifies the substrate binding modality. Y107A mutant enzyme displays a mechanism and rate constants for GSH binding approaching those of Alpha, Mu, and Pi isoenzymes. Comparison of available crystallographic data for all these GSTs reveals an unexpected evolutionary trend in terms of flexibility, which provides a basis for understanding our experimental results.
Pubblicato
Rilevanza internazionale
Articolo
Sì, ma tipo non specificato
Settore CHIM/07 - Fondamenti Chimici delle Tecnologie
Settore BIO/10
eng
Con Impact Factor ISI
Chemical bonds; Crystallography; Enzymes; Mutagenesis; Protons; Spectroscopic analysis; Substrates; Isoenzymes; Biochemistry; alanine; arginine; glutathione transferase; glutathione transferase t2 2; isoenzyme; unclassified drug; glutathione; GSTT2 protein, human; proton; thiol derivative; article; crystallography; enzyme activation; enzyme active site; enzyme binding; enzyme kinetics; evolution; potentiometry; priority journal; site directed mutagenesis; spectroscopy; chemical model; classification; comparative study; flow injection analysis; genetics; human; kinetics; metabolism; molecular evolution; mutation; pH; Arginine; Catalytic Domain; Evolution, Molecular; Flow Injection Analysis; Glutathione; Glutathione Transferase; Humans; Hydrogen-Ion Concentration; Isoenzymes; Kinetics; Models, Chemical; Mutagenesis, Site-Directed; Mutation; Protons; Sulfhydryl Compounds
Caccuri, A.M., Antonini, G., Board, P.G., Flanagan, J., Parker, M.W., Paolesse, R., et al. (2001). Human glutathione transferase T2-2 discloses some evolutionary strategies for optimization of substrate binding to the active site of glutathione transferases. THE JOURNAL OF BIOLOGICAL CHEMISTRY, 276(8), 5427-5431 [10.1074/jbc.M002819200].
Caccuri, Am; Antonini, G; Board, P; Flanagan, J; Parker, M; Paolesse, R; Turella, P; Federici, G; LO BELLO, M; Ricci, G
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2108/53599
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