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J Biol Chem, Vol. 273, Issue 36, 23267-23273, September 4, 1998

Flexibility of Helix 2 in the Human Glutathione Transferase P1-1
TIME-RESOLVED FLUORESCENCE SPECTROSCOPY

Lorenzo Stella, Anna Maria Caccuri^§, Nicola Rosato^¶, Maria Nicotra^§, Mario Lo Bello^§, Fabio De Matteis^**, Anna P. Mazzetti^§, Giorgio Federici, and Giorgio Ricci^§

From the Departments of  Chemical Sciences and Technologies, ^§ Biology, ^¶ Experimental Medicine and Biochemical Sciences, and  Physics, University of Rome "Tor Vergata," Via della Ricerca Scientifica 00133 Rome, Italy, the ^** Istituto Nazionale di Fisica della Materia, 00133 Rome, Italy, and the  Ospedale Pediatrico IRCCS "Bambin Gesú," 00165 Rome, Italy

Time-resolved fluorescence spectroscopy and site-directed mutagenesis have been used to probe the flexibility of -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-28 mutant enzyme was studied in which the second tryptophan of glutathione transferase P1-1 is replaced by histidine. Time-resolved fluorescence data indicate that, in the absence of glutathione, the apoenzyme exists in at least two different families of conformational states. The first one (38% of the total population) corresponds to a number of slightly 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 centered at 320 nm, thus suggesting a quite apolar environment near Trp-38. The interconversion between these two conformations is much slower than 1 ns. In the presence of saturating glutathione concentrations, the 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, and 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 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, 3028-3034) indicating the existence of a pre-complex in which GSH is not firmly bound to the active site.

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