FLUORESCENCE CHARACTERIZATION OF TRP-21 IN RAT GLUTATHIONE-S-TRANSFERASE 1-1 - MICROCONFORMATIONAL CHANGES INDUCED BY S-HEXYL GLUTATHIONE

The glutathione S-transferase (GST) isoenzyme Al-1 from rat contains a single tryptophan, Trp 21, which is expected to lie within alpha-heli x 1 based on comparison with the X-ray crystal structures of the pi- a nd mu-class enzymes. Steady-state and multifrequency phase/modulation fluorescence studies have been performed in order to characterize the fluorescence parameters of this tryptophan and to document ligand-indu ced conformational changes in this region of the protein. Addition of S-hexyl glutathione to GST isoenzyme A1-1 causes an increase in the st eady-state fluorescence intensity, whereas addition of the substrate g lutathione has no effect. Frequency-domain excited-state lifetime meas urements indicate that Trp 21 exhibits three exponential decays in sub strate-free GST. In the presence of S-hexyl glutathione, the data are also best described by the sum of three exponential decays, but the re covered lifetime values change. For the substrate-free protein, the sh ort lifetime component contributes 9-16% of the total intensity at fou r wavelengths spanning the emission. The fractional intensity of this lifetime component is decreased to less than 3% in the presence of S-h exyl glutathione. Steady-state quenching experiments indicate that Trp 21 is insensitive to quenching by iodide, but it is readily quenched by acrylamide. Acrylamide-quenching experiments at several emission wa velengths indicate that the long-wavelength components become quenched more easily in the presence of S-hexyl glutathione. Differential fluo rescence polarization measurements also have been performed, and the d ata describe the sum of two anisotropy decay rates. The recovered rota tional correlation times for this model are 26 ns and 0.81 ns, which c an be attributed to global motion of the protein dimer, and fast local motion of the tryptophan side chain. These results demonstrate that r egions of GST that are not in direct contact with bound substrates are mobile and undergo microconformational rearrangement when the ''H-sit e'' is occupied.