title: Efficient catalysis of disulfide formation during protein folding with a single active-site cysteine.
abstract: Protein disulfide isomerases (PDIs) catalyze disulfide bond formation during protein folding in vivo and are essential for viability in eukaryotic cells. They share the active-site sequence C-X-X-C that forms a catalytic disulfide. The recent finding that the EUG1 protein, a PDI-related yeast protein, with C-X-X-S sequence at its active sites can complement PDI-deficiency raised the general question of whether disulfide-isomerase activity is essential for cell viability or whether PDI variants with single active-site thiol groups can be catalytically active as disulfide isomerases. We investigated the function of the catalytic cysteine residues in DsbA, a PDI-related protein required for disulfide formation in the periplasmic space of Escherichia coli, by replacing C30 and C33 with alanine. While the mutant C30A and the double mutant CC30/33AA are inactive, C33A catalyzes disulfide-interchange reactions and oxidative renaturation of the reduced, unfolded thrombin inhibitor hirudin with close to wild-type efficiency. Thus, the single active-site thiol group of C30 is sufficient for disulfide-isomerase activity of the DsbA protein.
authors.affiliation: Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Wunderlich
authors.forename: A
authors.initials: A
authors.lastname: Otto
authors.forename: K
authors.initials: K
authors.lastname: Maskos
authors.forename: M
authors.initials: M
authors.lastname: Mücke
authors.forename: R
authors.initials: R
authors.lastname: Seckler
authors.forename: R
authors.initials: R
authors.lastname: Glockshuber
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D005978:Glutathione / Q000737:chemistry; D006629:Hirudins / Q000737:chemistry; D007535:Isomerases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding; D013329:Structure-Activity Relationship
chemical_list: D017931:DNA Primers; D004220:Disulfides; D006629:Hirudins; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione