Ligand Effects on the Fluorescence Properties of Tyrosine-9 in Alpha 1-1 Glutathione S-Transferase

Abstract

A conserved tyrosine plays a critical role in catalysis by mammalian glutathione S-transferases (GSTs) of the alpha-, mu-, and pi-classes, by forming a hydrogen bond to and stabilizing the thiolate form of glutathione. The hydrogen bonding properties of this tyrosine in the rat A1-1 GST (Tyr-9), in the absence and presence of ligands, have been studied by steady state and time-resolved fluorescence spectroscopy. In order to achieve this, the single tryptophan (Trp 21) found in the rat A1-1 GST has been replaced with the fluorometrically silent phenylalanine (W21F). Additionally, a double mutant lacking this tryptophan and the catalytic tyrosine (W21F:Y9F) has been constructed, and these mutants have been used as probes of ligand effects at Tyr-9. A comparison of the correlated excitation--emission spectra of the W21F mutant and the W21F-Y9F indicates that a red-shifted emission component is contributed by Tyr-9 with excitation bands at 255 and 300 nm, in the ligand-free enzyme. The pH-dependence of the intensity of these spectral cross-peaks is consistent with an active site tyrosine with a pKa of 8.1-8.3. Upon addition of GSH, the red-shifted component is quenched. Multifrequency phase/modulation fluorescence experiments qualitatively demonstrate that GSH causes a decrease in the average excited state lifetime on the red-edge of the spectrum of W21F but not of the W21F:Y9F spectrum. Steady state correlated difference spectra (W21F-W21F:Y9F) have been used to obtain a model for the excitation-emission correlated spectrum of Tyr-9, which indicates that Tyr-9 is heterogeneous at pH 7.5, with properties of both tyrosinate and "normal tyrosine". The tyrosinate fraction is eliminated, and the blue-shifted component becomes more intense upon addition of GSH conjugates, indicating that the weak hydrogen bond between Tyr-9 and thioethers has little charge-transfer character. The S-methyl GSH yields an "anomalous" spectrum at pH 7.5, which retains cross-peaks consistent with ionized tyrosinate. These results indicate that, in the absence of ligand, Tyr-9 forms a strongly polarized hydrogen bond or a fraction of the phenolic hydroxyl group is partially deprotonated. However, when a GSH conjugate with a sufficiently large hydrophobic group occupies the H-site, Tyr-9 is fully protonated, with little charge-transfer character.

Intrinsic Tyrosine Fluorescence as a Tool To Study the Interaction of the Shaker B “Ball” Peptide with Anionic Membranes

“…The triexponential behavior of the fluorescence decay observed in both peptides has been similarly reported for other peptides and proteins with a single tyrosine residue (16,30,31,43,44) and reflects the existence of groundstate rotamers sensing different chemical environments, which interconvert slowly on the nanosecond time scale (45,46). The small differences observed in the intermediate and longest lifetimes of ShB-L7E compared to those of ShB should correspond to differences in the local interactions sensed by two of the three rotamers due to the presence of the glutamic acid.…”

Investigation of the Anticoagulant Mechanisms of a Covalent Antithrombin-Heparin Complex

“…2). However, intrinsic fluorescence of ATH at lower wavelengths was slightly greater than that for noncovalent AT⅐SH complex; a result that may indicate a difference in the environment around tyrosyl residues in the conjugate (25,26). Alternatively, interactions between the AT and heparin of ATH at nontryptophanyl residues may be different from the corresponding noncovalent binding of AT and SH, leading to modified allosteric effects at ATH tryptophan 49 (27).…”

Ensemble Perspective for Catalytic Promiscuity

“…Construction of the rat GSTA1-1 (rGSTA1-1) ⌬209 -222 mutant has been described previously, as have the expression and purification (38,39). Activity of all purified enzymes was determined using the 1-chloro-2,4-dinitrobenzene (CDNB) assay (40).…”