SOLUTION STRUCTURE OF GLUTATHIONE BOUND TO HUMAN GLUTATHIONE TRANSFERASE P1-1 - COMPARISON OF NMR MEASUREMENTS WITH THE CRYSTAL-STRUCTURE

The conformation of the bound glutathione (GSH) in the active site of the human glutathione transferase P1-1 (EC 2.5.1.18) has been studied by transferred NOE measurements and compared with those obtained by X- ray diffraction data. Two-dimensional TRNOESY and TRROESY experiments have been performed under fast-exchange conditions. The family of GSH conformers, compatible with TRNOE distance constraints, shows a backbo ne structure very similar to the crystal model. Interesting difference s have been found in the side chain regions. After restrained energy m inimization of a representative NMR conformer in the active site, the sulfur atom is not found in hydrogen-bonding distance of the hydroxyl group of Tyr 7. This situation is similar to the one observed in an '' atypical'' crystal complex grown at low pH and low temperature. The NM R conformers display also a poorly defined structure of the glutamyl m oiety, and the presence of an unexpected intermolecular NOE could indi cate a different interaction of this substrate portion with the G-site . The NMR data seem to provide a snapshot of GSH in a precomplex where the GSH glutamyl end is bound in a different fashion. The existence o f this precomplex is supported by pre-steady-state kinetic experiments [Caccuri, A. M., Lo Bello, M., Nuccetelli, M., Nicotra, M., Rossi, P. , Antonini, G., Federici, G., and Ricci, G. (1998) Biochemistry 37, 30 28-3034] and preliminary time-resolved fluorescence data.