Selenoglutaredoxin as a glutathione peroxidase mimic.
Glutaredoxin (Grx1) from Escherichia coli is a monomeric, 85-amino-acid-long, disulfide-containing redox protein. A Grx1 variant in which the redox-active disulfide was replaced with a selenocysteine (C11U/C14S) was prepared by native chemical ligation from three fragments as a potential mimic of the natural selenoenzyme glutathione peroxidase (Gpx). Selenoglutaredoxin, like the analogous C14S Grx1 variant, shows weak peroxidase activity. The selenol provides a 30-fold advantage over the thiol, but its activity is four orders of magnitude lower than that of bovine Gpx. In contrast, selenoglutaredoxin is an excellent catalyst for thiol-disulfide exchange reactions; it promotes the reduction of beta-hydroxyethyldisulfide by glutathione with a specific activity of 130 units mg(-1). This value is 1.8 times greater than that of C14S Grx1 under identical conditions, and >10(4) greater than the peroxidase activity of either enzyme. Given the facile reduction of the glutathionyl-selenoglutaredoxin adduct by glutathione, oxidation of the selenol by the alkyl hydroperoxide substrate likely limits catalytic turnover and will have to be optimized to create more effective Gpx mimics. These results highlight the challenge of generating Gpx activity in a small, generic protein scaffold, despite the presence of a well-defined glutathione binding site and the intrinsic advantage of selenium over sulfur derivatives.
Laboratorium für Organische Chemie, ETH Zürich, Hönggerberg HCI F339, 8093 Zürich, Switzerland.
Giulio
G
Casi
Gerard
G
Roelfes
Donald
D
Hilvert
D000595:Amino Acid Sequence; D002384:Catalysis; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D054477:Glutaredoxins / Q000737:chemistry / Q000378:metabolism*; D005979:Glutathione Peroxidase / Q000378:metabolism*; D007700:Kinetics; D010446:Peptide Fragments; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012643:Selenium; D017279:Selenocysteine / Q000378:metabolism
D029968:Escherichia coli Proteins; D054477:Glutaredoxins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D017279:Selenocysteine; D005979:Glutathione Peroxidase; D012643:Selenium