title: pH profiles indicative of rate-limiting nucleophilic displacement in thioltransferase catalysis.
abstract: The apparent pKa 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 displays an inflection point near pH 8.5. The similarity of the pH-rate profile with the titration of the GSH thiol moiety suggested rate-limiting nucleophilic attack by the glutathionyl thiolate species to regenerate reduced TTase from the TTase-SSG intermediate. To test this hypothesis pH-rate profiles for TTase-catalyzed dethiolation of the glutathionyl mixed disulfide of bovine serum albumin ([35S]BSA-SSG) were measured according to release of radiolabeled GS-equivalents. Various thiol compounds, whose thiol pKa values range on both sides of the pKa of GSH (pKa = 8.7), were used as reducing substrates, e.g., trifluoroethanethiol (pKa = 7.5) and 3-mercaptopropionic acid (pKa = 10.3). The pH-rate profiles paralleled the titration of the respective thiol groups of the reducing substrates, consistent with the hypothesis. In addition, second-order rate constants (k) were determined for the nonenzymatic and TTase-catalyzed reactions of the various thiols with BSA-SSG. A simple linear free energy relationship (log k vs pKa) was displayed for the nonenzymatic reactions. In contrast, the relationship for the enzymatic reactions revealed GSH to be different from the other thiol substrates, i.e., GSH gave a second-order rate constant greater than expected for its thiol pKa. This result suggests a special interaction of GSH with the TTase enzyme in the transition state that enhances the nucleophilicity of GSH.
authors.affiliation: Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4965, USA.
authors.forename: U
authors.initials: U
authors.lastname: Srinivasan
authors.forename: P A
authors.initials: PA
authors.lastname: Mieyal
authors.forename: J J
authors.initials: JJ
authors.lastname: Mieyal
mesh_terms: D000818:Animals; D002384:Catalysis; D002417:Cattle; D054477:Glutaredoxins; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008962:Models, Theoretical; D010088:Oxidoreductases / Q000378:metabolism*; D011490:Protein Disulfide Reductase (Glutathione); D012710:Serum Albumin, Bovine; D013379:Substrate Specificity
chemical_list: D054477:Glutaredoxins; D012710:Serum Albumin, Bovine; D010088:Oxidoreductases; D011490:Protein Disulfide Reductase (Glutathione); D005978:Glutathione