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Ribonucleotide reductase (RNR) from Lactobacillus leichmannii, a 76 kDa monomer using adenosylcobalamin (AdoCbl) as a cofactor, catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is rapidly (&lt;30 s) inactivated by 1 equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F(2)CTP). [1'-(3)H]- and [5-(3)H]F(2)CTP were synthesized and used independently to inactivate RNR. Sephadex G-50 chromatography of the inactivation mixture revealed that 0.47 equiv of a sugar was covalently bound to RNR and that 0.71 equiv of cytosine was released. Alternatively, analysis of the inactivated RNR by SDS-PAGE without boiling resulted in 33% of RNR migrating as a 110 kDa protein. Inactivation of RNR with a mixture of [1'-(3)H]F(2)CTP and [1'-(2)H]F(2)CTP followed by reduction with NaBH(4), alkylation with iodoacetamide, trypsin digestion, and HPLC separation of the resulting peptides allowed isolation and identification by MALDI-TOF mass spectrometry (MS) of a (3)H/(2)H-labeled peptide containing C(731) and C(736) from the C-terminus of RNR accounting for 10% of the labeled protein. The MS analysis also revealed that the two cysteines were cross-linked to a furanone species derived from the sugar of F(2)CTP. Incubation of [1'-(3)H]F(2)CTP with C119S-RNR resulted in 0.3 equiv of sugar being covalently bound to the protein, and incubation with NaBH(4) subsequent to inactivation resulted in trapping of 2'-fluoro-2'-deoxycytidine. These studies and the ones in the preceding paper (DOI: 10.1021/bi9021318 ) allow proposal of a mechanism of inactivation of RNR by F(2)CTP involving multiple reaction pathways. The proposed mechanisms share many common features with F(2)CDP inactivation of the class I RNRs.

Inactivation of Lactobacillus leichmannii Ribonucleotide Reductase by 2′,2′-Difluoro-2′-deoxycytidine 5′-Triphosphate: Covalent Modification