THE ROLE OF THE BURIED ASPARTATE OF ESCHERICHIA-COLI THIOREDOXIN IN THE ACTIVATION OF THE MIXED DISULFIDE INTERMEDIATE

The structurally homologous protein disulfide isomerases and thioredox ins exhibit a 10(5) variation of redox equilibria. It is demonstrated that the kinetic distinction among these protein family members lies p rimarily 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 rats of Escherichia coli thioredoxi n by dithiothreitol is directly proportional to the fraction of Asp-26 in the protonated farm over the pH range of 6-9. The kinetic role of Asp-26 is further probed via differential solvent kinetic isotope effe ct measurements versus a D26N variant, The differential solvent isotop e effect of 0.6 is consistent with a direct proton donation to the thi olate leaving group (Cys-35) via an enforced general acid catalysis by trapping mechanism, Such a donation necessitates a structural rearran gement as these two buried side chains are separated by 6 Angstrom in both the oxidized and reduced forms of the protein.