Efficient catalysis of disulfide formation during protein folding with a single active-site cysteine.
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.
Wunderlich
M
M
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Germany.
Otto
A
A
Maskos
K
K
Mücke
M
M
Seckler
R
R
Glockshuber
R
R
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
D017931:DNA Primers; D004220:Disulfides; D006629:Hirudins; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione