PROTON RELEASE UPON GLUTATHIONE BINDING TO GLUTATHIONE TRANSFERASE P1-1 - KINETIC-ANALYSIS OF A MULTISTEP GLUTATHIONE BINDING PROCESS

The fate of the thiol proton coming from the ionization of the sulfhyd ryl group of GSH in the active site of glutathione transferase P1-1 ha s been studied. pH changes caused by the binding of GSH to the enzyme in the absence of any inorganic buffer indicate that the thiol proton leaves the active site when the binary complex is formed. The amount o f protons released is stoichiometric to the amount of GSH thiolate for med in the G-site. The apparent pK(a) value for the bound GSH, calcula ted with this potentiometric approach, is 6.18 +/- 0.09; very similar values are found by spectrophotometric (6.20 +/- 0.12) and by kinetic (6.00 +/- 0.08) experiments. Binding of S-hexylglutathione does not ca use any proton release. Stopped-flow data obtained by means of an acid -base indicator show that the proton extrusion process (apparent t(1/2 ) = 1.1 +/- 0.1 ms at 15 degrees C) is not rate limiting in turnover ( apparent t(1/2) = 34 +/- 4 ms at 15 degrees C). By comparing the kinet ic behavior of three distinct events occurring during the binding of G SH to the enzyme, i.e., proton release, ionization of bound GSH and qu enching of intrinsic fluorescence, it appears that the binding process follows a multistep mechanism possibly involving the conformational t ransition of a weak precomplex into the final Michaelis complex. This step is modulated by helix 2 motions and may be rate limiting at physi ological GSH concentrations. These findings, coming from kinetic studi es, are consistent with NMR data [Nicotra, M., Paci, M., Sette, M., Oa kley, A. J., Parker, M. W., Lo Bello, M., Caccuri, A. M., Federici, G. , and Ricci, G. (1998) Biochemistry 37, 3020-3027] and time-resolved f luorescence experiments [Stella, L., Caccuri, A. M., Rosato, N., Nicot ra, M., Lo Bello, M., De Matteis, F., Mazzetti, A. P., Federici, G., a nd Ricci, G., manuscript in preparation].