Ribonucleotide reductase (RNR, 76 kDa) from Lactobacillus leichmannii is a class II RNR that requires adenosylcobalamin (AdoCbl) as a cofactor. It catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is 100% inactivated by 1 equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F2CTP) in < 2 min. Sephadex G-50 chromatography of the inactivation reaction mixture for 2 min revealed that 0.47 equiv of a sugar moiety is covalently bound to RNR and 0.25 equiv of a cobalt(III) corrin is tightly associated, likely through a covalent interaction with C419 (Co-S) in the active site of RNR [Lohman, G. J. S., and Stubbe, J. (2010) Biochemistry 49, DOI: 10.1021/bi902132u]. After 1 h, a similar experiment revealed 0.45 equiv of the Co-S adduct associated with the protein. Thus, at least two pathways are associated with RNR inactivation: one associated with alkylation on by the sugar Of F2CTP and the second with AdoCbl destruction. To determine the fate of [1'-3H]F2CTP in the latter pathway, the reaction mixture at 2 min was reduced with NaB2H 4 (NaB2H4) and the protein separated from the small molecules using a centrifugation device. The small molecules were dephosphorylated and analyzed by HPLC to reveal 0.25 equiv of a stereoisomer of cytidine, characterized by mass spectrometry and NMR spectroscopy, indicating the trapped nucleotide had lost both of its fluorides and gained an oxygen. High-field ENDOR studies with [I'- 2H]F2CTP from the reaction quenched at 30 s revealed a radical that is nucleotide-based. The relationship between this radical and the trapped cytidine analogue provides insight into the nonalkylative pathway for RNR inactivation relative to the alkylative pathway.
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