Class II Ribonucleotide Reductases Catalyze Carbon−Cobalt Bond Reformation on Every Turnover

Ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the reduction of nucleotides to deoxynucleotides with concomitant oxidation of two cysteines within the active site to a disulfide. RTPR requires adenosylcobalamin (AdoCbl) as a cofactor and as a radical chain initiator. Catalysis is initiated by homolysis of the carbon−cobalt bond of AdoCbl to yield cob(II)alamin, 5‘-deoxyadenosine, and a protein-based thiyl radical. The turnover numbers for ATP with its allosteric effector dGTP, and for CTP with its allosteric effector dATP, are both 2 s - 1 . The rate-limiting step for turnover in the steady state is re-reduction of the oxidized form of the protein, a conformational change, or both. Under conditions where [RTPR] ≫ [AdoCbl], the rates of ATP and CTP reduction do not vary linearly with [AdoCbl] but instead exhibit saturation behavior with turnover numbers of 10 s - 1 (ATP) and 8.5 s - 1 (CTP). This result suggests that dissociation of AdoCbl, which requires carbon−cobalt bond reformation, follows nucleotide reduction, but precedes the rate-limiting step in catalysis. A presteady-state analysis of the ATP reduction (using rapid chemical quench methods) in the presence of [5‘- 3 H]-AdoCbl reveals formation of product dATP at a k obs of 55 ± 10 s - 1 and tritium washout from [5‘- 3 H]-AdoCbl at 0.6 s - 1 . The rate of washout is approximately equivalent to the rate of washout of 3 H in the absence of substrate. Measurement of the ratio of 3 H 2 O:dATP over time reveals that washout of 3 H occurs at the end of each turnover. Production of 3 H 2 O requires reformation of the carbon−cobalt bond. These steady-state and presteady-state data suggest that carbon−cobalt bond reformation and dissociation of AdoCbl into solution accompany each turnover and that the radical chain length of the RTPR-catalyzed nucleotide reduction is approximately one.