SCANNING AND ESCAPE DURING PROTEIN-DISULFIDE ISOMERASE-ASSISTED PROTEIN-FOLDING

During oxidative protein folding, efficient catalysis of disulfide rea rrangements by protein-disulfide isomerase is found to involve an esca pe mechanism that prevents the enzyme hom becoming trapped in covalent complexes with substrates that fail to rearrange in a timely fashion. Protein-disulfide isomerase mutants with only a single active-site cy steine catalyze slow disulfide rearrangements and become trapped in a covalent complex with substrate. Escape is mediated by the second, mor e carboxyl-terminal cysteine at the active site. A glutathione redox b uffer increases the k(cat) for single-cysteine mutants by 20-40-fold, but the presence of the second cysteine at the active site in the wild -type enzyme increases the k(cat) by over 200-fold. A model is develop ed in which kinetic scanning for disulfides of increasing reactivity i s timed against an intramolecular clock provided by the second cystein e at the active site. This provides an alternative, more efficient mec hanism for rearrangement involving the reduction and reoxidation of su bstrate disulfides.