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

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journal contribution
posted on 23.02.2010, 00:00 by Gregory J. S. Lohman, JoAnne Stubbe
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 (<30 s) inactivated by 1 equiv of 2′,2′-difluoro-2′-deoxycytidine 5′-triphosphate (F2CTP). [1′-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 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′-3H]F2CTP and [1′-2H]F2CTP followed by reduction with NaBH4, alkylation with iodoacetamide, trypsin digestion, and HPLC separation of the resulting peptides allowed isolation and identification by MALDI-TOF mass spectrometry (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 cysteines were cross-linked to a furanone species derived from the sugar of F2CTP. Incubation of [1′-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 studies and the ones in the preceding paper (DOI: 10.1021/bi9021318) allow proposal of a mechanism of inactivation of RNR by F2CTP involving multiple reaction pathways. The proposed mechanisms share many common features with F2CDP inactivation of the class I RNRs.

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