Ribonucleotide Reductase from Euglena gracilis, a Deoxyadenosylcobalamin-dependent Enzyme

A ribonucleotide reductase requiring 5'-deoxyadenosylcobalamin has been detected in Euglena gracilis var. bacillaris by measuring 3H exchange from labeled coenzyme to water. Extracts were partially purified by centrifugation, dialysis, and treatment with MnCl2. These extracts catalyzed the reduction of ribonucleoside triphosphates to the corresponding deoxyribonucleotides as measured by colorimetric assay with the diphenylamine procedure. The enzyme required 5'-deoxyadenosylcobalamin, a dithiol, and a ribonucleoside triphosphate for exchange activity and reduction. Monothiols could not replace the dithiol requirement. Ribonucleoside diphosphates were slightly active as substrates in both assay systems. Mg++, at low concentrations, did not affect the reaction; at concentrations greater than 2 mm, this ion was inhibitory. The reduction of ribonucleotides was inhibited by adding deoxyribonucleotides other than the immediate products; e.g. ATP reduction was inhibited by adding dGTP, dCTP, or dUTP, but not by dATP. dTTP inhibited the reduction of all ribonucleotides to the greatest extent. The molecular weight of this enzyme, determined by sucrose density gradient centrifugation, was approximately 145,000. Extracts of Ochromonas malhamensis and Ochromonas danica showed no activity in the 3H exchange assay under similar conditions.