Studies on Thioredoxin Reductase from <em>Escherichia coil</em> B.

Amino acid analyses on thioredoxin reductase indicate the following amino acid composition: Asp65Thr43Ser26Glu60Pro17Gly63Ala58Val32Met12Ileu39Leu53Tyr16Phe18Try2Lys23His18Arg29 (total half-cys)8 (CONH2)59. The enzyme consists of two identical or very similar polypeptide chains joined by noncovalent bonds. This is indicated from experiments employing ultracentrifugation in urea or guanidine hydrochloride, from the analysis of tryptic peptide maps and from quantitative determinations of NH2-terminal amino acids. Carboxymethylation of the enzyme with iodoacetic acid under different conditions showed that each polypeptide chain of the native enzyme contains one disulfide and two free sulfhydryl groups, the latter buried in the enzyme structure. Titration of the enzyme with NADPH under anaerobic conditions demonstrated that the complete reduction of one molecule of enzyme requires four molecules of NADPH. The results indicate that the enzyme contains two redox acceptors in addition to the two molecules of flavin adenine dinucleotide. Evidence is presented that these redox acceptors are identical with the two disulfides of the enzyme. Spectral studies of thioredoxin reductase employing anaerobic titration with substrate did not reveal the formation of a stable red 2-electron intermediate similar to that observed for two other flavoproteins, lipoyl dehydrogenase and glutathione reductase. This finding suggests that the catalytic mechanism for thioredoxin reductase is different from that of lipoyl dehydrogenase and glutathione reductase. [ABSTRACT FROM AUTHOR]