Proton Release upon Glutathione Binding to Glutathione Transferase P1-1: Kinetic Analysis of a Multistep Glutathione Binding Process
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- 作者：Anna Maria Caccuri ; Mario Lo Bello ; Marzia Nuccetelli ; Maria Nicotra ; Paola Rossi ; Giovanni Antonini ; Giorgio Federici ; and Giorgio Ricci
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：March 3, 1998
- 年：1998
- 卷：37
- 期：9
- 页码：3028 - 3034
- 全文大小：117K
- 年卷期：v.37,no.9(March 3, 1998)
- ISSN：1520-4995
文摘
The fate of the thiol proton coming from the ionization of thesulfhydryl group of GSH in theactive site of glutathione transferase P1-1 has been studied. pHchanges caused by the binding of GSHto the enzyme in the absence of any inorganic buffer indicate that thethiol proton leaves the active sitewhen the binary complex is formed. The amount of protons releasedis stoichiometric to the amount ofGSH thiolate formed in the G-site. The apparentpKa value for the bound GSH, calculated withthispotentiometric approach, is 6.18 ± 0.09; very similar values arefound by spectrophotometric (6.20 ±0.12) and by kinetic (6.00 ± 0.08) experiments. Binding ofS-hexylglutathione does not cause any protonrelease. Stopped-flow data obtained by means of an acid-baseindicator show that the proton extrusionprocess (apparent t1/2 = 1.1 ± 0.1 ms at15 
C) is not rate limiting in turnover (apparentt1/2 = 34 ± 4ms at 15 C). By comparing the kinetic behavior of threedistinct events occurring during the binding ofGSH to the enzyme, i.e., proton release, ionization of bound GSH andquenching of intrinsic fluorescence,it appears that the binding process follows a multistep mechanismpossibly involving the conformationaltransition of a weak precomplex into the final Michaelis complex.This step is modulated by helix 2motions and may be rate limiting at physiological GSH concentrations.These findings, coming fromkinetic 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] andtime-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].
C) is not rate limiting in turnover (apparentt1/2 = 34 ± 4ms at 15 C). By comparing the kinetic behavior of threedistinct events occurring during the binding ofGSH to the enzyme, i.e., proton release, ionization of bound GSH andquenching of intrinsic fluorescence,it appears that the binding process follows a multistep mechanismpossibly involving the conformationaltransition of a weak precomplex into the final Michaelis complex.This step is modulated by helix 2motions and may be rate limiting at physiological GSH concentrations.These findings, coming fromkinetic 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] andtime-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].