PH PROFILES INDICATIVE OF RATE-LIMITING NUCLEOPHILIC DISPLACEMENT IN THIOLTRANSFERASE CATALYSIS

The apparent pK(a) for the active site thiol of human thioltransferase (TTase) is about 3.5, but the pH dependence of TTase-catalyzed rates of glutathione (GSH)-dependent reduction of disulfide substrates displ ays an inflection point near pH 8.5. The similarity of the pH-rate pro file with the titration of the GSH thiol moiety suggested rate-limitin g nucleophilic attack by the glutathionyl thiolate species to regenera te reduced TTase from the TTase-SSG intermediate. To test this hypothe sis pH-rate profiles for TTase-catalyzed dethiolation of the glutathio nyl mixed disulfide of bovine serum albumin ([S-35]BSA-SSG) were measu red according to release of radiolabeled GS-equivalents. Various thiol compounds, whose thiol pK(a) values range on both sides of the pK(a) of GSH (pK(a) = 8.7), were used as reducing substrates, e.g., trifluor oethanethiol (pK(a) = 7.5) and 3-mercaptopropionic acid (pK(a) = 10.3) . The pH-rate profiles paralleled the titration of the respective thio l groups of the reducing substrates, consistent with the hypothesis. I n addition, second-order rate constants (k) were determined for the no nenzymatic and TTase-catalyzed reactions of the various thiols with BS A-SSG. A simple linear free energy relationship (log k vs pK(a)) was d isplayed for the nonenzymatic reactions. In contrast, the relationship for the enzymatic reactions revealed GSH to be different from the oth er thiol substrates, i.e., GSH gave a second-order rate constant great er than expected for its thiol pK(a). This result suggests a special i nteraction of GSH with the TTase enzyme in the transition state that e nhances the nucleophilicity of GSH.