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 sulfhydryl group of GSH in the active site of glutathione transferase P1-1 has 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 of protons released is stoichiometric to the amount of GSH thiolate formed in the G-site. The apparent pKa value for the bound GSH, calculated 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 cause any proton release. Stopped-flow data obtained by means of an acid-base indicator show that the proton extrusion process (apparent t1/2 = 1.1 +/- 0.1 ms at 15 degrees C) is not rate limiting in turnover (apparent t1/2 = 34 +/- 4 ms at 15 degrees C). By comparing the kinetic behavior of three distinct events occurring during the binding of GSH to the enzyme, i. e., proton release, ionization of bound GSH and quenching of intrinsic fluorescence, it appears that the binding process follows a multistep mechanism possibly involving the conformational transition of a weak precomplex into the final Michaelis complex. This step is modulated by helix 2 motions and may be rate limiting at physiological GSH concentrations. These findings, coming from kinetic studies, are consistent with NMR data [Nicotra, M., Paci, M., Sette, M., Oakley, A. J., Parker, M. W., Lo Bello, M., Caccuri, A. M., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3020-3027] and time-resolved fluorescence experiments [Stella, L., Caccuri, A. M., Rosato, N., Nicotra, M., Lo Bello, M., De Matteis, F., Mazzetti, A. P., Federici, G., and Ricci, G., manuscript in preparation].
Caccuri
A M
AM
Department of Biology, University of Rome "Tor Vergata", Rome, Italy.
Lo Bello
M
M
Nuccetelli
M
M
Nicotra
M
M
Rossi
P
P
Antonini
G
G
Federici
G
G
Ricci
G
G
D005978:Glutathione / Q000378:metabolism*; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D010920:Placenta / Q000201:enzymology; D011487:Protein Conformation; D011522:Protons; D013050:Spectrometry, Fluorescence
D007527:Isoenzymes; D011522:Protons; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase; D005978:Glutathione