Cobamides and Ribonucleotide Reduction

When deoxyadenosylcobalamin was incubated with ribonucleoside triphosphate reductase from Lactobacillus leichmannii, dihydrolipoate, and a nucleoside triphosphate in the presence of tritiated water, labeled hydrogen was incorporated into nonexchangeable positions in the cobamide. A maximum of 1.4 atoms of labeled hydrogen was incorporated per molecule of deoxyadenosylcobalamin. On photolysis of the labeled cobamide the radioactivity was found in the 5'-hydrogen atoms of the nucleoside products derived from the deoxyadenosyl moiety. This result indicates that the hydrogen atoms of the methylene group bound to cobalt in the cobamide exchange with water, a conclusion confirmed by nonenzymic synthesis of 5-deoxyadenosylcobalamin-5'-3H2 and the demonstration that tritium is transferred from this compound into water. This hydrogen exchange occurred only in the presence of enzyme, dithiol, and a nucleotide. The dithiol requirement could be met by 1,3- or 1,4-dithiols or by the thioredoxin system, but monothiols were relatively ineffective. Ribonucleoside triphosphates could satisfy the nucleotide requirement, but deoxyribonucleoside triphosphates were also active and dGTP was the most effective nucleotide of all. Nucleoside mono- and diphosphates were less effective than the corresponding triphosphates. The specificity of the nucleotide requirement, the absence of hydrogen transfer to or from the activating nucleotide, and the fact that deoxynucleotides did not undergo detectable reaction in the system suggest that nucleotides act as allosteric activators of the hydrogen exchange reaction. The enzymic transfer of hydrogen between water and the 5'-methylene group of the cobamide is considered to occur via the dithiol and to indicate the reversible formation of a reduced cobamide intermediate, probably identical with that occurring in the mutase reactions that require deoxyadenosylcobalamin and in the diol dehydrase reaction. In ribonucleotide reduction this reduced cobamide intermediate is regarded as the immediate donor of a hydride ion to position 2' α of the nucleotide substrate. Attempts to obtain evidence of the existence of this intermediate by incorporating label from deoxyadenosylcobalamin-5'-3H2 into the deoxynucleotide product of the reductase or by dilution by unlabeled deoxyadenosylcobalamin of tritium incorporation from water into deoxynucleotide product were unsuccessful. However, as required by the hypothesis, inactivation of the enzyme for the hydrogen transfer reaction under various conditions always resulted in parallel or greater inactivation for ribonucleotide reduction.