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.

The Dithiol Glutaredoxins of African Trypanosomes Have Distinct Roles and Are Closely Linked to the Unique Trypanothione Metabolism

“…Interestingly, in E. coli Grx2, which has the highest activity of the three bacterial dithiol Grxs toward 2-ME-SSG, the Lys is replaced by a Tyr. This supports previous reports that the characteristic ␥-glutamate moiety is more important for GSH recognition than the glycine part (43,44). In T. brucei Grx1 and Grx2, these substitutions probably support the interaction with T(SH) 2 (see below), in which the glycine carboxylate does not carry a negative charge but is engaged in an amide bond with the spermidine bridge.…”

Mentha piperita as a pivotal neuro-protective agent against gamma irradiation induced DNA fragmentation and apoptosis

“…GSH serves as an electron donor for glutathione peroxidase, GSH also contributes to the detoxification and elimination of toxic electrophilic metabolites and xenobiotics (Commandeur et al 1995). GSH selectively recycles oxidized glutaredoxin to the reduced enzyme, generating GSSG, which in turn is reduced to GSH by GR (Srinivasan et al 1997). Furthermore, the beneficial effect of pretreatment with Mentha on radiation induced tissue injury, cell cycle arrest, and apoptosis may be related to the effectiveness of Mentha against chromosomal damage and/or may be through improvement of DNA content by the antimutagenic properties of Mentha piperita (Samman et al 1998).…”

Targeted disruption of the glutaredoxin 1 gene does not sensitize adult mice to tissue injury induced by ischemia/reperfusion and hyperoxia

“…[ 35 S]BSA-SSG was prepared as described previously with the following modifications [38]. S-carboxymethyl BSA was initially reacted with N-succinimidylpyridyl bis (3,3'-dithio-propionate) and the reaction was then quenched with glycine.…”