Steady state kinetics measurements performed on human placenta glutathione transferase (GST P 1-1), utilizing 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) as co-substrate, show that the kcat value (approximately equal to 1.2 s-1) is pH-independent between pH 4.0 and 7.0 and is scarcely affected by the nature of the leaving group. The pH profile of kcat/KmNBD-Cl suggests a pKa > or = 6.0 for GSH bound to the enzyme. Pre-steady state experiments demonstrate the presence of a burst-phase in which the conjugation product (or the sigma-complex intermediate) accumulates in an amount stoichiometric to the GST active site concentration. These results indicate that the steady state kinetics of GST P 1-1 with NBD-Cl are independent of the deprotonation of the bound GSH between pH 4.0 and 7.0 because the rate-limiting step is the product release. The occurrence of a fast enzymatic conjugation of GSH with a number of poor substrates or even electrophilic inhibitors of GST, mainly performed in a single turnover reaction, may reveal a further detoxicating role of GST.

Caccuri, A.m., Ascenzi, P., LO BELLO, M., Federici, G., Battistoni, A., Mazzetti, P., et al. (1994). Are the steady state kinetics of glutathione transferase always dependent on the deprotonation of the bound glutathione? New insights in the kinetic mechanism of GST P 1-1. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 200(3), 1428-1434 [10.1006/bbrc.1994.1610].

Are the steady state kinetics of glutathione transferase always dependent on the deprotonation of the bound glutathione? New insights in the kinetic mechanism of GST P 1-1

CACCURI, ANNA MARIA;LO BELLO, MARIO;FEDERICI, GIORGIO;BATTISTONI, ANDREA;RICCI, GIORGIO
1994-05-16

Abstract

Steady state kinetics measurements performed on human placenta glutathione transferase (GST P 1-1), utilizing 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) as co-substrate, show that the kcat value (approximately equal to 1.2 s-1) is pH-independent between pH 4.0 and 7.0 and is scarcely affected by the nature of the leaving group. The pH profile of kcat/KmNBD-Cl suggests a pKa > or = 6.0 for GSH bound to the enzyme. Pre-steady state experiments demonstrate the presence of a burst-phase in which the conjugation product (or the sigma-complex intermediate) accumulates in an amount stoichiometric to the GST active site concentration. These results indicate that the steady state kinetics of GST P 1-1 with NBD-Cl are independent of the deprotonation of the bound GSH between pH 4.0 and 7.0 because the rate-limiting step is the product release. The occurrence of a fast enzymatic conjugation of GSH with a number of poor substrates or even electrophilic inhibitors of GST, mainly performed in a single turnover reaction, may reveal a further detoxicating role of GST.
16-mag-1994
Pubblicato
Rilevanza internazionale
Articolo
Esperti anonimi
Settore BIO/10 - BIOCHIMICA
English
Con Impact Factor ISI
oxidation-reduction; glutathione; kinetics; hydrogen-ion concentration; humans; 4-chloro-7-nitrobenzofurazan; glutathione transferase
Caccuri, A.m., Ascenzi, P., LO BELLO, M., Federici, G., Battistoni, A., Mazzetti, P., et al. (1994). Are the steady state kinetics of glutathione transferase always dependent on the deprotonation of the bound glutathione? New insights in the kinetic mechanism of GST P 1-1. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 200(3), 1428-1434 [10.1006/bbrc.1994.1610].
Caccuri, Am; Ascenzi, P; LO BELLO, M; Federici, G; Battistoni, A; Mazzetti, P; Ricci, G
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/69116
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