Mechanism of B12-dependent ribonucleotide reductase.

Abstract

Ribonucleotide reductase from L. leichmmannii catalyzes cleavage of the carbon cobalt bond of AdoCbl homolytically in a kinetically competent fashion. This cleavage triggers a chain of events which results in cleavage of the 3'C-H bond of the nucleotide substrate followed by cleavage of the 2' carbon hydroxyl bond. Involvement of a radical cation has been suggested as a possible mechanism by which this unusual reduction reaction might occur. Furthermore, cleavage of the 3' carbon hydrogen bond of [3'-3H]NTP resulted in no 3H release to solvent and no 3H recovered in AdoCbl. These results were interpreted to mean that in this system AdoCbl does not serve as a H abstractor, but rather as a radical chain initiator. A protein residue on the RTPR is postulated to carry out the actual H abstraction from the substrate. These results and the conclusions drawn from them are further supported by recent experiments using [3'-3H]ClUTP. Incubation of RTPR with [3'-3H]ClUTP resulted in release of 3H2O, uracil, PPPi, formation of CoII and 5' deoxyadenosine. The 3H2O release confirms the enzyme's ability to cleave the 3'C-H bond of a nucleotide analog. Furthermore, little if any 3H was recovered in the 5' deoxyadenosine and the rate of 3H2O release from [3'3H]ClUTP was 12 times faster than the rate of 3H2O release from [5'-3H]AdoCbl. These observations support the conclusions drawn from data with the normal substrate; ie, AdoCbl serves as a radical chain initiator rather than a direct H abstractor from substrate.