Steady state, pre-steady state kinetic experiments, and site-directed mutagenesis have been used to dissect the catalytic mechanism of human glutathione transferase T2-2 with 1-menaphthyl sulfate as co-substrate, This enzyme is close to the ancestral precursor of the more recently evolved glutathione transferases belonging to Alpha, Pi, and Mu classes. The enzyme displays a random kinetic mechanism with very low k(cat) and k(cat)/ K-m(GSH) values and with a rate-limiting step identified as the product release. The chemical step, which is fast and causes product accumulation before the steady state catalysis, strictly depends on the deprotonation of the bound GSH. Replacement of Arg-107 with Ala dramatically affects the fast phase, indicating that this residue is crucial both in the activation and orientation of GSH in the ternary complex. All pre-steady state and steady state kinetic data were convincingly fit to a kinetic mechanism that reflects a quite primordial catalytic efficiency of this enzyme. It involves two slowly interconverting or not interconverting enzyme populations (or active sites of the dimeric enzyme) both able to bind and activate GSH and strongly inhibited by the product. Only one population or subunit is catalytically competent. The proposed mechanism accounts for the apparent half-site behavior of this enzyme and for the apparent negative cooperativity observed under steady state conditions. These findings also suggest some evolutionary strategies in the glutathione transferase family that have been adopted for the optimization of the catalytic activity, which are mainly based on an increased flexibility of critical protein segments and on an optimal orientation of the substrate.

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 the catalytic activity of glutathione transferases. THE JOURNAL OF BIOLOGICAL CHEMISTRY, 276(8), 5432-5437 [10.1074/jbc.M002818200].

Human glutathione transferase T2-2 discloses some evolutionary strategies for optimization of the catalytic activity of glutathione transferases

CACCURI, ANNA MARIA;PAOLESSE, ROBERTO;RICCI, GIORGIO
2001

Abstract

Steady state, pre-steady state kinetic experiments, and site-directed mutagenesis have been used to dissect the catalytic mechanism of human glutathione transferase T2-2 with 1-menaphthyl sulfate as co-substrate, This enzyme is close to the ancestral precursor of the more recently evolved glutathione transferases belonging to Alpha, Pi, and Mu classes. The enzyme displays a random kinetic mechanism with very low k(cat) and k(cat)/ K-m(GSH) values and with a rate-limiting step identified as the product release. The chemical step, which is fast and causes product accumulation before the steady state catalysis, strictly depends on the deprotonation of the bound GSH. Replacement of Arg-107 with Ala dramatically affects the fast phase, indicating that this residue is crucial both in the activation and orientation of GSH in the ternary complex. All pre-steady state and steady state kinetic data were convincingly fit to a kinetic mechanism that reflects a quite primordial catalytic efficiency of this enzyme. It involves two slowly interconverting or not interconverting enzyme populations (or active sites of the dimeric enzyme) both able to bind and activate GSH and strongly inhibited by the product. Only one population or subunit is catalytically competent. The proposed mechanism accounts for the apparent half-site behavior of this enzyme and for the apparent negative cooperativity observed under steady state conditions. These findings also suggest some evolutionary strategies in the glutathione transferase family that have been adopted for the optimization of the catalytic activity, which are mainly based on an increased flexibility of critical protein segments and on an optimal orientation of the substrate.
Pubblicato
Rilevanza internazionale
Articolo
Sì, ma tipo non specificato
Settore BIO/10
Settore CHIM/07 - Fondamenti Chimici delle Tecnologie
English
Con Impact Factor ISI
Catalysis; Dimers; Enzymes; Mutagenesis; pH effects; Substrates; Random kinetic mechanism; Biochemistry; 1 menaphthyl sulfate; alanine; arginine; enzyme precursor; glutathione transferase; glutathione transferase t2 2; sulfate; unclassified drug; 1-menaphthyl sulfate; glutathione; GSTT2 protein, human; isoenzyme; naphthalene derivative; article; catalysis; enzyme binding; enzyme kinetics; enzyme mechanism; enzyme release; molecular evolution; priority journal; proton transport; site directed mutagenesis; steady state; chemical model; genetics; human; kinetics; metabolism; Felis catus; Arginine; Catalysis; Evolution, Molecular; Glutathione; Glutathione Transferase; Humans; Isoenzymes; Kinetics; Models, Chemical; Naphthalenes
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 the catalytic activity of glutathione transferases. THE JOURNAL OF BIOLOGICAL CHEMISTRY, 276(8), 5432-5437 [10.1074/jbc.M002818200].
Caccuri, Am; Antonini, G; Board, P; Flanagan, J; Parker, M; Paolesse, R; Turella, P; Chelvanayagam, G; Ricci, G
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2108/44556
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