Identification of the catalytic site of rat liver glutathione peroxidase as selenocysteine

• Published in: Biochemistry (Easton) Vol. 17; no. 13; pp. 2639 - 2644
• Main Authors: Forstrom, John W, Zakowski, Jack J, Tappel, Al L
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 27.06.1978
• Subjects: Amino Acids - analysis; Animals; Binding Sites; Cysteine - analogs & derivatives; Glutathione Peroxidase - isolation & purification; Glutathione Peroxidase - metabolism; Liver - enzymology; Mass Spectrometry; Peroxidases - metabolism; Rats; Selenium
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00606a028

A procedure was developed to isolate 75Se-labeled rat liver glutathione peroxidase (glutathione:H2O2 oxidoreductase, EC 1.11.1.9) at 30--50% purity with 20--30% yields in 4--5 days. Using these preparations of glutathione peroxidase, the selenium moiety in the enzyme was identified as selenocysteine by derivatizing the seleno group with either iodoacetate or ethylenimine in the intact protein, hydrolyzing the protein with 6 N HCl, and cochromatographing the 75Se-labeled products with known standards. Techniques employed were anion-exchange chromatography, cation-exchange chromatography, gel-permeation chromatography, two-dimensional thin-layer chromatography, and automated amino acid analysis. The selenocysteine moiety was identified as the catalytic site in glutathione peroxidase by specifically labeling the enzyme with [14C]iodoacetate on the 75Se-labeled selenium atom and fractionating the 14C, 75Se-labeled derivative after acid hydrolysis. It was concluded that the reduced form of glutathione peroxidase contains the selenocysteine selenol (-SeH) at the catalytic site.