The amino acid sequence of membrane-associated prostaglandin (PG) E synthase-2 (mPGE synthase-2), which has a broad specificity in its thiol requirement for a catalytic activity, has the consensus region from (104)Leu to (120)Leu found in glutaredoxin and of thioredoxin. The sequence of Cys-x-x-Cys in the consensus region is the active site for thioredoxin and mPGE synthase-2 also has this amino acid sequence ((110)Cys-x-x-(113)Cys). The mutation from (110)Cys to Ser or the double mutation from (110)Cys and (113)Cys to Ser caused loss of PGE synthase activity, whereas the single mutation from (110)Cys to Ser did not affect the enzyme activity. These results indicate that (110)Cys, but not (113)Cys, is the essential amino acid in the active site of mPGE synthase-2. (110)Cys is an important amino acid in PGE synthase activity and plays the critical role as Cys at the same position in redoxin. Moreover, we found that the reduced form of lipoic acid (dihydrolipoic acid) serves as one of the natural activators of mPGE synthase-2 in the cells. (C) 2003 Elsevier Science (USA). All rights reserved.