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.

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.