title: Scanning and escape during protein-disulfide isomerase-assisted protein folding.
abstract: During oxidative protein folding, efficient catalysis of disulfide rearrangements by protein-disulfide isomerase is found to involve an escape mechanism that prevents the enzyme from 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 cysteine catalyze slow disulfide rearrangements and become trapped in a covalent complex with substrate. Escape is mediated by the second, more carboxyl-terminal cysteine at the active site. A glutathione redox buffer increases the kcat 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 kcat by over 200-fold. A model is developed in which kinetic scanning for disulfides of increasing reactivity is timed against an intramolecular clock provided by the second cysteine at the active site. This provides an alternative, more efficient mechanism for rearrangement involving the reduction and reoxidation of substrate disulfides.
authors.affiliation: Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030, USA.
authors.forename: K W
authors.initials: KW
authors.lastname: Walker
authors.forename: H F
authors.initials: HF
authors.lastname: Gilbert
mesh_terms: D000818:Animals; D001665:Binding Sites; D002021:Buffers; D002417:Cattle; D004229:Dithiothreitol / Q000494:pharmacology; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D019803:Glutathione Disulfide; D007535:Isomerases / Q000378:metabolism*; D007536:Isomerism; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases; D051381:Rats; D012260:Ribonucleases / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology
chemical_list: D002021:Buffers; D013439:Sulfhydryl Reagents; D012260:Ribonucleases; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D004229:Dithiothreitol; D019803:Glutathione Disulfide