Thioredoxin reductase (TR) catalyzes the NADPH dependent reduction of the redox-active disulfide bond of thioredoxin (Trx). In mammals and other higher eukaryotes, TR contains the rare amino acid selenocysteine at the active site. The mitochondrial TR from C. elegans however contains a standard cysteine residue at its active site in place of selenocysteine. In order to characterize the catalytic properties of the enzyme, the C. elegans TR was cloned from an expressed sequence tag and then expressed in E. coli as an intein-fusion protein. This construct also carries a chitin binding domain that allows for affinity purification. The dimeric enzyme was purified by loading cellular extract onto an affinity column and then cleaving TR from the intein by addition of thiol to the buffer. The purified enzyme has a kcat of 665 min-1 and a Km of 700 µM using E. coli thioredoxin as substrate. This is about 30% of the activity demonstrated by the mammalian enzyme and is 550 fold higher than a cysteine mutant of mammalian TR. The enzyme would reduce selenocystine, but not hydrogen peroxide. The results indicate that the enzyme is an unusually reactive TR that contains a reactive cysteine residue in place of the usual selenocysteine residue. Selenocysteine is thus not a chemical necessity for the catalytic reaction. The mitochondrial TR from C. elegans is thus optimized to use sulfur in place of selenium, and probably increases the reactivity of the active-site cysteine by use of an enzymic base.
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