Fluorescence characterization of Trp 21 in rat glutathione S‐transferase 1–1: Microconformational changes induced by S‐hexyl glutathione

Abstract

The glutathione S-transferase (GST) isoenzyme Al-1 from rat contains a single tryptophan, Trp 21, which is expected to lie within a-helix 1 based on comparison with the X-ray crystal structures of the pi-and 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-induced conformational changes in this region of the protein. Addition of S-hexyl glutathione to GST isoenzyme Al-1 causes an increase in the steady-state fluorescence intensity, whereas addition of the substrate glutathione has no effect. Frequencydomain excited-state lifetime measurements indicate that Trp 21 exhibits three exponential decays in substratefree GST. In the presence of S-hexyl glutathione, the data are also best described by the sum of three exponential decays, but the recovered lifetime values change. For the substrate-free protein, the short lifetime component contributes 9-16070 of the total intensity at four wavelengths spanning the emission. The fractional intensity of this lifetime component is decreased to less than 3% in the presence of S-hexyl 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 wavelengths indicate that the long-wavelength components become quenched more easily in the presence of S-hexyl glutathione. Differential fluorescence polarization measurements also have been performed, and the data describe the sum of two anisotropy decay rates. The recovered rotational correlation times for this model are 26 ns and 0.81 ns, which can be attributed to global motion of the protein dimer, and fast local motion of the tryptophan side chain. These results demonstrate that regions of GST that are not in direct contact with bound substrates are mobile and undergo microconformational rearrangement when the "H-site" is occupied.

Monobromobimane as an Affinity Label of the Xenobiotic Binding Site of Rat Glutathione S-Transferase 3-3

“…The 330-nm emission is characteristic of tryptophan residues in a hydrophobic environment as is observed for rat glutathione S-transferase 1-1 (39). The 460-nm emission is the result of the incorporated bimane moiety (20).…”

The Anomalous pK of Tyr-9 in Glutathione S-Transferase A1-1 Catalyzes Product Release

“…The binding of S-hexylglutathione (S-hexyl-GSH) to GSTA1-1 has been shown to induce closure of the C terminus, with Tyr-9 pK a (ϳ9) remaining lower than the pK a of free tyrosine in solution (13,24,25). Here the emission spectra of W21F/F222W and Y9F/W21F/F222W were also obtained in the presence of saturating concentrations of S-hexyl-GSH.…”

Flexibility of Helix 2 in the Human Glutathione Transferase P1-1

“…Furthermore, the existence of different conformations that interconvert in times slower than the fluorescence lifetime results in a non-exponential intensity decay, and thus can be detected. Recently, time-resolved fluorescence spectroscopy has been applied to study glutathione S-transferase A1-1 from rat (21); since this isoenzyme has a single Trp per subunit, located in the H-site, the intrinsic fluorescence was used to characterize the dynamics of that region. Another fluorescence study focused on the protonation state of Tyr-7 of the same isoenzyme (22).…”