SUBSTITUTING SELENOCYSTEINE FOR ACTIVE-SITE CYSTEINE 149 OF PHOSPHORYLATING GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE REVEALS A PEROXIDASE-ACTIVITY

Replacing the essential Cys-149 by a selenocysteine into the active si te of phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus leads to a selenoGAPDH that mimics a selenoperoxidase activity. Saturation kinetics were observed with cum enyl and tert-butyl. hydroperoxides, with a better catalytic efficienc y for the aromatic compound. The enzymatic mechanism fits a sequential model where the formation of a ternary complex between the holoseleno enzyme, the 3-carboxy 4-nitrobenzenethiol used as the reductant and th e hydroperoxide precedes product release. The fact that the selenoGAPD H is NAD-saturated supports a binding of hydroperoxide and reductant i n the substrate binding site. The catalytic efficiency is similar to s elenosubtilisins but remains low compared to selenoglutathione peroxid ase. This is discussed in relation to what is known from the X-ray cry stal structures of selenoglutathione peroxidase and GAPDHs. (C) 1998 F ederation of European Biochemical Societies.