FLEXIBILITY OF HELIX-2 IN THE HUMAN GLUTATHIONE TRANSFERASE P1-1 - TIME-RESOLVED FLUORESCENCE SPECTROSCOPY

Time-resolved fluorescence spectroscopy and site-directed mutagenesis have been used to probe the flexibility of alpha-helix 2 (residues 35- 46) in the apo structure of the human glutathione transferase P1-1 (EC 2.5.1.18) as well as in the binary complex with the natural substrate glutathione. Trp-38, which resides on helix 2, has been exploited as an intrinsic fluorescent probe of the dynamics of this region. A Trp-2 8 mutant enzyme was studied in which the second tryptophan of glutathi one transferase P1-1 is replaced by histidine. Time-resolved fluoresce nce data indicate that, in the absence of glutathione, the apoenzyme e xists in at least two different families of conformational states. The first one (38% of the total population) corresponds to a number of sl ightly different conformations of helix 2, in which Trp-38 resides in a polar environment showing an average emission wavelength of 350 nm. The second one (62% of the total population) displays an emission cent ered at 320 nm, thus suggesting a quite apolar environment near Trp-38 . The interconversion between these two conformations is much slower t han 1 ns. In the presence of saturating glutathione concentrations, th e equilibrium is shifted toward the apolar component, which is now 83% of the total population. The polar conformers, on the other hand, do not change their average decay lifetime, but the distribution becomes wider, indicating a slightly increased rigidity. These data suggest a central role of conformational transitions in the binding mechanism, a nd are consistent with NMR data (Nicotra, M., Paci, M., Sette, M., Oak ley, A. J., Parker, M. W., Lo Bello, M., Caccuri, A. M., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3020-3027) and pre-steady state kinetic experiments (Caccuri, A. M., Lo Bello, M., Nuccetelli, M., Ni cotra, M., Rossi, P., Antonini, G., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3028-3034) indicating the existence of a pre-complex in which GSH is not firmly bound to the active site.