Enzymatic Synthesis of Deoxyribonucleotides. Iv. Isolation and Characterization of Thioredoxin, the Hydrogen Donor from Escherichia Coli B.

The reductive formation of deoxycytidine diphosphate from cytidine diphosphate with crude extracts of Escherichia coli B required the presence of reduced triphosphopyridine nucleotide (1). On purification of the GDP-reductase system, an enzyme fraction (Fraction B) was obtained, which was no longer active with TPNH but, instead, showed an absolute requirement for reduced lipoate (2, 3). Several lines of evidence suggested, however, that reduced lipoate acted as a model substance in the purified CDP-reductase system, and that the physiological hydrogen donor was removed during the purification of Fraction B. We have now purified from E. coli 13 a low molecular weight, heat-stable protein, hereafter called thioredoxin’ With bacterial Fraction 13, the requirement for reduced lipoate could be replaced by either (a) catalytic amounts of thioredoxin + TPNH or (b) substrate amounts of chemically reduced dihydrothioredoxin. It was also found that E. coli B (and purified Fraction B) contained an enzyme (thioredoxin reductasel), which catalyzed the reduction of thioredoxin by TPNH. This reaction involved the transformation of a disulfide group to a disulfhydryl group according to Equation 1: