THE CATALYTIC MECHANISM OF GLUTATHIONE-S-TRANSFERASE (GST) - SPECTROSCOPIC DETERMINATION OF THE PK(A) OF TYR-9 IN RAT ALPHA-1-1 GST

The rat alpha1-1 glutathione S-transferase (GST) contains a single, no n-essential tryptophan and only 8 tyrosines in each subunit. One of th ese tyrosines, Tyr-9, hydrogen bonds to the substrate glutathione and stabilizes the nucleophilic thiolate anion. Two mutant proteins that a llow for the spectroscopic determination of the pK(a) of this catalyti c residue have been constructed. The W21F mutant provides a fully acti ve GST with no tryptophans, and the double mutant W21F/Y9F lacks both tryptophan and the active site tyrosine. The intrinsic fluorescence an d absorbance properties of these mutants are dominated by tyrosine. Fl uorescence emission, fluorescence excitation, and absorbance spectral changes of samples containing the W21F mutant at several pH values in the range 6.8-9.0 reveal a pH-dependent increase in the contribution o f tyrosinate. No spectral changes are observed with the W21F/Y9F prote in in this pH range. At pH 12.5, both proteins exhibit complete deprot onation of all tyrosines. The pK(a) of Tyr-9 determined from these spe ctroscopic changes is 8.3-8.5. The changes in absorbance at 250 and 29 5 nm correspond to titration of 0.95 +/- 0.29 tyrosines/subunit in the W21F protein between pH 6.9 and 9.3. Moreover, addition of the inhibi tor S-hexyl-glutathione results in an apparent increase in the pK(a) o f Tyr-9. Together, these results indicate that the catalytically activ e Tyr of GSTs has a pK(a) value that is 1.8-2.0 pK(a) units below tyro sine in solution. It is likely that this decrease in the pK(a) of Tyr- 9 contributes to catalysis by altering the equilibrium position of the proton shared between Tyr-9 and GSH, and this active site residue may function as a general base catalyst in addition to a hydrogen bond do nor.