The Selenium Moiety of Glutathione Peroxidase

Summary

Glutathione peroxidase (E.C.1.11.1.9) the tetrametric enzyme which contains 4 atoms of selenium, was purified to homogeneity from bovine red cells and investigated by means of chemical modification and ultraviolet difference spectroscopy. The borohydride-reduced enzyme was alkylated with labeled haloacetates and the stoichiometry of binding was determined.

Chloroacetate turned out to be a suitable active-site-directed reagent. Consequently, the reduced enzyme was carboxymethylated using 1-[¹⁴C]-chloroacetate, hydrolyzed, and subjected to amino acid analysis. Distribution of radioactivity and selenium resulted in a pattern similar to that obtained with analogously prepared carboxymethyl-seleno-cysteine.

The difference spectrum of substrate-reduced against oxidized GSH-peroxidase shows a distinct peak at 237 nm which is attributed to four cysteine residues per mol of enzyme. In the presence of urea, which reversibly inactivated the enzyme, KBH₄-reduced and GSH-reduced enzyme yield a difference spectrum with a sharp peak at 248 nm, corresponding to a selenol content of four groups per tetramer.

Taken together, the present findings demonstrate that the selenium moiety of GSH-peroxidase consists of a compound which can be reduced to seleno-cysteine by KBH₄ but not by GSH. In addition, a cysteine residue seems to be involved in catalysis.