The role of the buried aspartate of Escherichia coli thioredoxin in the activation of the mixed disulfide intermediate

Abstract

The structurally homologous protein disulfide isomerases and thioredoxins exhibit a 105 variation of redox equilibria. It is demonstrated that the kinetic distinction among these protein family members lies primarily in the rate of breakdown of the mixed disulfide intermediate. The conserved buried acid group serves as a proton transfer catalyst for the buried active site cysteine in the formation and breakdown of the mixed disulfide. The reduction rate of Escherichia coli thioredoxin by dithiothreitol is directly proportional to the fraction of Asp-26 in the protonated form over the pH range of 6-9. The kinetic role of Asp-26 is further probed via differential solvent kinetic isotope effect measurements versus a D26N variant. The differential solvent isotope effect of 0.6 is consistent with a direct proton donation to the thiolate leaving group (Cys- 35) via an enforced general acid catalysis by trapping mechanism. Such a donation necessitates a structural rearrangement as these two buried side chains are separated by 6 Å in both the oxidized and reduced forms of the protein.