title: 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.
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.
authors.affiliation: Department of Biology, University of Rome Tor Vergata, Italy.
authors.forename: A M
authors.initials: AM
authors.lastname: Caccuri
authors.forename: P
authors.initials: P
authors.lastname: Ascenzi
authors.forename: M
authors.initials: M
authors.lastname: Lo Bello
authors.forename: G
authors.initials: G
authors.lastname: Federici
authors.forename: A
authors.initials: A
authors.lastname: Battistoni
authors.forename: P
authors.initials: P
authors.lastname: Mazzetti
authors.forename: G
authors.initials: G
authors.lastname: Ricci
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000737:chemistry; D005978:Glutathione / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction
chemical_list: D005982:Glutathione Transferase; D009327:4-Chloro-7-nitrobenzofurazan; D005978:Glutathione