Thiol ester hydrolysis catalyzed by glutathione S-transferase A1-1.

rGSTA1-1 has been shown to catalyze the hydrolysis of the thiol ester glutathionyl ethacrynate (E-SG). In contrast, neither the retro-Michael addition with the substrate EA-SG, to yield GSH and ethacrynic acid (EA), nor the conjugation reaction between GSH and EA to yield the thiol ester E-SG was catalyzed to any measurable extent under similar conditions. The steady state kcat and KM for hydrolysis of E-SG by wild type rGSTA1-1 were 0.11 +/- 0.009 min-1 and 15.7 +/- 1.6 mM, respectively. The site-directed mutant, Y9F, in which the catalytic Tyr-9 is substituted with Phe, was completely inactive in this reaction. To uncover a mechanistic signature that would distinguish between direct hydrolysis and covalent catalysis involving acylation of Tyr-9, solvent isotope exchange and mass spectrometry experiments were performed. No 18O incorporation into the starting thiol ester was detected with initial velocity solvent isotope exchange experiments. However, covalent adducts corresponding to acylated protein also were not observed by electrospray ionization mass spectrometry, even with an assay that minimized the experimental dead time and which allowed for detection of N-acetyltyrosine acylated with EA in a chemical model system. The kon and koff rate constants for association and dissociation of E-SG were determined, by stopped flow fluorescence, to be 5 x 10(5) s-1 M-1 and 6.7 s-1, respectively. Together with the isotope partitioning results, these rate constants were used to construct partial free energy profiles for the GST-catalyzed hydrolysis of E-SG, assuming that Tyr-9 acts as a general acid-base catalyst. The "one-way flux" of the thiol esterase reaction results directly from the thermodynamic stability of the products after rate-limiting attack of the thiol ester by H2O or Tyr-9, and is sufficient to drive the hydrolysis to completion, in contrast to GST-catalyzed breakdown of other GSH conjugates.