1-Cys Peroxiredoxin, a Bifunctional Enzyme with Glutathione Peroxidase and Phospholipase A2 Activities

This report provides definitive evidence that the protein 1-Cys peroxiredoxin is a bifunctional (“moonlighting”) enzyme with two distinct active sites. We have previously shown that human, rat, and bovine lungs contain an acidic Ca 2+ -independent phospholipase A 2 (aiPLA 2 ). The cDNA encoding aiPLA 2  was found to be identical to that of a non-selenium glutathione peroxidase (NSGPx). Protein expressed using a previously reported E. coli construct which has a His-tag and 50 additional amino acids at the NH 2 terminus, did not exhibit aiPLA 2 activity. A new construct which contains the His-tag plus two extra amino acids at the COOH terminus when expressed in Escherichia coli generated a protein that hydrolyzed the sn -2 acyl chain of phospholipids at pH 4, and exhibited NSGPx activity with H 2 O 2 at pH 8. The expressed 1-Cys peroxiredoxin has identical functional properties to the native lung enzyme: aiPLA 2 activity is inhibited by the serine protease inhibitor, diethyl p -nitrophenyl phosphate, by the tetrahedral mimic 1-hexadecyl-3-trifluoroethylglycero- sn -2-phosphomethanol (MJ33), and by 1-Cys peroxiredoxin monoclonal antibody (mAb) 8H11 but these agents have no effect on NSGPx activity; NSGPx activity is inhibited by mercaptosuccinate and by 1-Cys peroxiredoxin mAb 8B3 antibody which have no effect on aiPLA 2 activity. Mutation of Ser 32 to Ala abolishes aiPLA 2 activity, yet the NSGPx activity remains unaffected; a Cys 47 to Ser mutant is devoid of peroxidase activity but aiPLA 2 activity remains intact. These results suggest that Ser 32 in the GDSWG consensus sequence provides the catalytic nucleophile for the hydrolase activity of aiPLA 2 , while Cys 47 in the PVCTTE consensus sequence is at the active site for peroxidase activity. The bifunctional catalytic properties of 1-Cys peroxiredoxin are compatible with a simultaneous role for the protein in the regulation of phospholipid turnover as well as in protection against oxidative injury.