Inactivation of Lactobacillus leichmannii ribonucleotide reductase by 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate: covalent modification

Abstract

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 (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.

FIGURE 1

HPLC purification of peptides from RTPR inactivated with [1′-³H]-F₂CTP, treated with NaBH₄ at 2 min, alkylated with iodoacetamide and digested with trypsin. A_214nm (—), cpm (- - -). (A) Initial purification, linear gradient (^……) 0.1% TFA, 0–45% CH₃CN over 90 min. Region I, 50–53 min, 18.1% of radioactivity. Region II, 54–59 min, 19.7% of radioactivity. Region III, 60–63 min, 13.9% of radioactivity. Region IV, 63–67 min, 26% of radioactivity. (B) repurification of region I, linear gradient (^……) 10 mM NH₄OAc, pH 6.8, 0–35% CH₃CN over 90 min. 80% recovery of radiolabel.

FIGURE 2

Full MALDI-TOF analysis of the peptides isolated in Region I from the trypsin digest of RTPR inactivated with F₂CTP and treated with NaBH₄. (A) [1′-³H]-F₂CTP, NaBH₄. (B) [1′-²H,³H]-F₂CTP, NaBH₄. (C) [1′-³H]-F₂CTP, NaBD₄. The features at 1102.5 Da and 1347.7 Da are not from RTPR, as MS/MS fragmentation analysis showed no matches with sequences from RTPR.. Inset shows a zoom of (A) around the 2004 peak to highlight the presence of a peak at 2020 Da.

FIGURE 3

(A) Expansion of the MALDI spectrum in Figure 2 in the region around the 2004 Da peak. (I) [1′-³H]-F₂CTP, NaBH₄. (II) [1′-²H,³H]-F₂CTP, NaBH₄. (III) [1′-³H]-F₂CTP, NaBD₄. (B) Expansion of the MALDI spectrum in Figure 2 in the region around the 2063 Da peak. (I) [1′-³H]-F₂CTP, NaBH₄. (II) [1′-²H,³H]-F₂CTP, NaBH₄. (III) [1′-³H]-F₂CTP, NaBD₄.

FIGURE 4

(A) MS/MS of peak at 2063 Da, containing a label derived from F₂CTP. The y series is marked, and corresponds to the C-terminal peptide of RTPR (DLELVDQTDCEGGACPIK). The absolute MWs detected are shifted +43 Da relative to the acetamide labeled C-terminal peptide (Figure S1), suggesting alkylation at cysteine by a group 43Da larger than acetamide. The modification is present to some extent on each cysteine, as indicated by the appearance of the y series of the normal peptide after C₇₃₁ is cleaved (indicated at 831, 702, 645, 588 and 517 Da).

FIGURE 5

MS/MS of the peak at 2004 Da corresponding to the C-terminal peptide of RTPR (DLELVDQTDCEGGACPIK) containing an internal cysteine-cysteine crosslink derived from F₂CTP. The y ion series is indicated. No regular ions were detected in the mass range 360–970 Da.

FIGURE 6

SDS PAGE of RTPR inactivated by treatment with F₂CTP.

SCHEME 1

Proposed mechanism of inactivation of RNR by 2′-substituted mechanism based inhibitors.

SCHEME 2

Proposal for structures that can be accommodated by the labeling observed in the MS data.

SCHEME 3

Proposed mechanism of inactivation by RTPR by F₂CTP by the non-alkylative and alkylative pathways.

SCHEME 4

Proposed mechanism for generation of furanone during the inactivation of RTPR with F₂CTP.