Inactivation of Lactobacillus leichmannii Ribonucleotide Reductase by 2′,2′-Difluoro-2′-deoxycytidine 5′-Triphosphate: Covalent Modification
journal contributionposted 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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110 kDa proteinMS analysisDOIC 731C 736RNR accountingHPLC separationreaction pathwaysC 119Sinactivation mixture0.3 equiv0.47 equivLactobacillus leichmannii76 kDa monomernucleoside triphosphatesCovalent ModificationRibonucleotide reductaseF 2CDP inactivationfuranone speciesNaBH 4mechanisms shareF 2CTP Incubationtrypsin digestionLactobacillus leichmannii Ribonucleotide ReductaseF 2CTP0.71 equiv1 equivSDS