LIGAND EFFECTS ON THE FLUORESCENCE PROPERTIES OF TYROSINE-9 IN ALPHA-1-1-GLUTATHIONE-S-TRANSFERASE

A conserved tyrosine plays a critical role in catalysis by mammalian g lutathione S-transferases (GSTs) of the alpha-, mu-, and pi-classes, b y forming a hydrogen bond to and stabilizing the thiolate form of glut athione. The hydrogen bonding properties of this tyrosine in the rat A 1-1 GST (Tyr-9), in the absence and presence of ligands, have been stu died by steady state and time-resolved fluorescence spectroscopy. In o rder to achieve this, the single tryptophan (Trp 21) found in the rat A1-1 GST has been replaced with the fluorometrically silent phenylalan ine (W21F). Additionally, a double mutant lacking this tryptophan and the catalytic tyrosine (W21F:Y9F) has been constructed, and these muta nts have been used as probes of ligand effects at Tyr-9. A comparison of the correlated excitation-emission spectra of the W21F mutant and t he W21F-Y9F indicates that a red-shifted emission component is contrib uted by Tyr-9 with excitation bands at 255 and 300 nm, in the ligand-f ree enzyme. The pH-dependence of the intensity of these spectral cross -peaks is consistent with an active site tyrosine with a pK(a) of 8.1- 8.3. Upon addition of GSH, the red-shifted component is quenched. Mult ifrequency phase/modulation fluorescence experiments qualitatively dem onstrate that GSH causes a decrease in the average excited state lifet ime on the red-edge of the spectrum of W21F but not of the W21F:Y9F sp ectrum. Steady state correlated difference spectra (W21F - W21F:Y9F) h ave 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 t yrosinate fraction is eliminated, and the blue-shifted component becom es more intense upon addition of GSH conjugates, indicating that the w eak hydrogen bond between Tyr-9 and thioethers has little charge-trans fer character. The S-methyl GSH yields an ''anomalous'' spectrum at pH 7.5, which retains cross-peaks consistent with ionized tyrosinate. Th ese results indicate that, in the absence of ligand, Tyr-9 forms a str ongly polarized hydrogen bond or a fraction of the phenolic hydroxyl g roup is partially deprotonated. However, when a GSH conjugate with a s ufficiently large hydrophobic group occupies the H-site, Tyr-9 is full y protonated, with little charge-transfer character.