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

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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‘-3H]-AdoCbl reveals formation of product dATP at a kobs of 55 ± 10 s-1 and tritium washout from [5‘-3H]-AdoCbl at 0.6 s-1. The rate of washout is approximately equivalent to the rate of washout of 3H in the absence of substrate. Measurement of the ratio of 3H2O:dATP over time reveals that washout of 3H occurs at the end of each turnover. Production of 3H2O 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.