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

Ribonucleotide reductase (RNR) from Lactobacillus leichmannii, a 76 kDa monomer using adenosyleobalamin (AdoCbl) as a cofactor, catalyzes the conversion of nucleoside triphosphates to deoxynuclèotides and is rapidly (<30 s) inactivated by I equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F2CTP). [l-3H]. and [5-3H]F2CTP 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 RN Rand that 0.71 equiv of eytosine 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 mixlure of [I-3H]F2CTPand [l-2HIF2CTP followed by reduction with NaBH4, alkylation with iodoaeetamide, trypsin digestion, and HPLC separation of the resulting peptides allowed isolation and identification by MALDI-TOF mass speetrometry (MS) of a 3H/2H-labeled peptide containing C731 and C736 from the C-terminus of RNR accounting for 10% of the labeled protein. The MS analysis also revealed that the two eysteines were cross-linked to a furanone species derived from the sugar of F2CTP. Incubation of [l'-3H]F2CTP with C119S-RNR resulted in 0.3 equiv of sugar being covalently bound to the protein, and incubation with NaBH4 subsequent to inactivation resulted in trapping of 2'-fluoro-2'-deoxycytidine. These sludieS and the ones in the preceding paper (DOI: l0.1021/bi9021318) allow proposal of a mechanism of inactivation of RN R by F2CTP involving multiple reaction pathways. The proposed mechanisms share many common features with F2CDP inactivation of the class I RNRs. [ABSTRACT FROM AUTHOR]