Dm. Lemaster et al., THE ROLE OF THE BURIED ASPARTATE OF ESCHERICHIA-COLI THIOREDOXIN IN THE ACTIVATION OF THE MIXED DISULFIDE INTERMEDIATE, The Journal of biological chemistry, 272(48), 1997, pp. 29998-30001
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.