Glutathione-dependent Synthesis of Deoxyribonucleotides

This reaction in vitro requires the combined function of three pure Escherichia coli proteins, namely the Bl and B2 subunits of ribonucleotide reductase and glutaredoxin. The stoichiometry was demonstrated by determinations of the products r3H]dCDP and GSSG. The standard assay couples oxidation of NADPH to the reduction of GSSG by glutathione reductase. The apparent K,,, value of glutaredoxin with ribonucleotide reductase at 4 mM GSH was 1.3 x 10e7 M. The molecular activity of glutaredoxin in CDP reduction was about lo-fold higher than that of thioredoxin and similar to the corresponding values for the Bl and B2 subunits of ribonucleotide reductase. The apparent V,, values in vitro of ribonucleotide reductase with the thioredoxin or glutaredoxin systems were similar. The apparent K,,, value for GSH was 0.40 mM in the presence of excess NADPH, glutathione reductase, and CDP. The dithiol or reduced form of glutaredoxin, isolated after chemical reduction with dithiothreitol, reduced a stoichiometric amount of CDP to dCDP in the presence of ribonucleotide reductase. In the absence of NADPH and glutathione reductase, the velocity of the reduction of CDP to dCDP by GSH was highly influenced by the ratio of GSH to GSSG and even small amounts of GSSG inhibited strongly. Inhibition by GSSG is a possible physiological control mechanism for deoxyribonucleotide synthesis.