Inactivation of Lactobacillus leichmannii ribonucleotide reductase by F2CTP: adenosylcobalamin destruction and formation of a nucleotide based radical

Ribonucleotide reductase (RNR, 76 kDa) from Lactobacillus leichmannii is a class II RNR that requires adenosylcobalamin (AdoCbl) as a cofactor. It catalyzes the conversion on nucleoside triphosphates to deoxynucleotides and is 100% inactivated by one equivalent (eq.) of 2′,2′difluoro-2′-deoxycytidine 5′-triphosphate (F2CTP) in <2 min. Sephadex G50 chromatography of the inactivation reaction for 2 min revealed that 0.47 eq. of a sugar moiety are covalently bound to RNR and 0.25 eq. of a cobalt III corrin are tightly associated, likely through a covalent interaction with C419 (Co-S) in the active site of RNR (Lohman et. al. (2009) Biochemistry, accompanying manuscript). After one hour, a similar experiment revealed 0.45 eq. of the Co-S adduct associated with the protein. Thus at least two pathways are associated with RNR inactivation: one associated with alkylation by the sugar of F2CTP and the second with AdoCbl destruction. To determine the fate of [1′-3H] F2CTP in the latter pathway, the reaction mixture at 2 min was reduced with NaBH4 (NaBH4) and the protein separated from the small molecules using a centrifugation device. The small molecules were dephosphorylated and analyzed by HPLC to reveal 0.25 eq. of a stereoisomer of cytidine, characterized by mass spectrometry and NMR spectroscopy, indicating the trapped nucleotide had lost both of its fluorides and gained an oxygen. High field ENDOR studies with [1′-2H] F2CTP from the reaction quenched at 30 s, revealed a radical that is nucleotide based. The relationship of this radical to the trapped cytidine analog provides insight into the non-alkylative pathway for RNR inactivation relative to the alkylative pathway. Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides providing the monomeric precursors required for DNA replication and repair.(1–5) Class I and Class II RNRs are stoichiometrically inactivated by the 5′-di and triphosphate forms of 2′deoxy-2′,2′-difluorocytidine (GemzarTM, F2C) a drug presently used clinically in the treatment of advanced pancreatic cancer and non small cell lung carcinoma.(6–11) The Lactobacillus leichmannii ribonucleoside triphosphate reductase, RTPR, a monomer of molecular weight 76 kDa, is the paradigm for adenosylcobalamin (AdoCbl) requiring RNRs, although recent §Funding for this study was provided in part by NIH grant GM-29595. *To whom correspondence should be addressed. Tel: (617) 253-1814, Fax: (617) 258-7247, [email protected]. SUPPORTING INFORMATION AVAILABLE UV-vis spectra of cobalamin standards (Figure 1S) and small molecule fraction from Sephadex G50 chromatography of the inactivation experiment (Figure 2S). 130 GHz EPR spectra of RTPR with F2CTP and [1′-2H]-F2CTP (Figure 3S). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author Manuscript Biochemistry. Author manuscript; available in PMC 2011 February 23. Published in final edited form as: Biochemistry. 2010 February 23; 49(7): 1396–1403. doi:10.1021/bi9021318. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript genomic analyses have revealed that the dimeric class II enzymes, exemplified by the recently crystallized Thermotoga maritima RTPR, are much more prevalent than the monomeric forms. (12–15) In the accompanying paper we reported the synthesis of [1′-3H]-, [1′-2H]-, and [5-H]-F2CTP and showed that one equivalent (eq.) of [1′-H]-F2CTP resulted in 90% inhibition of the enzyme within 30 seconds with 0.47 eq. of 3H covalently attached to the enzyme.(16) Our earlier studies demonstrated that during this inactivation, on a 30 min time scale, that RTPR became covalently labeled with the corrin ring of AdoCbl through C419, one of the active site cysteines providing reducing equivalents to generate dNTPs.(17) In the present paper we describe our efforts to examine the fate of AdoCbl immediately subsequent to enzyme inactivation and the fate of the remaining F2CTP that is not covalently attached to the enzyme. As with many mechanism based inhibitors of RNRs, multiple modes of inhibition are observed. (1,18) A model to accommodate our observations described in this paper in relationship to the observations in the accompanying manuscript is presented (Scheme 1). MATERIALS AND METHODS Quantification and characterization of cobalamin, cytosine, and nucleotide products generated from RTPR inactivated by F2CTP The inactivation mixture in final volume of 1250 μL contained: pre-reduced RTPR (50 μM), dATP (500 μM), AdoCbl (50 μM), HEPES (25 mM, pH 7.5), EDTA (4 mM), and MgCl2 (1 mM). After addition of AdoCbl, all aliquots were handled under red light and wrapped with foil. The inactivation was initiated by addition of either [1′-H]-F2CTP (specific activity (SA) 1985 cpm/nmol) or [5-H]-F2CTP (SA 1350 cpm/nmol) to a final concentration of 50 μM. An aliquot was assayed for activity as described in the accompanying paper.(16) The inactivation was allowed to proceed for either 2 min or 1 h at 37°C. An aliquot (100 μL) was removed after 2 min and after 1 h, quenched by filtration through a YM-30 membrane at 4°C, the nucleotides dephosphorylated with alkaline phosphatase and analyzed by HPLC. An aliquot of 1000 μL after 2 min and 1 h was loaded on a Sephadex G-50 column (1 × 20 cm, 20 mL) wrapped in foil, run at 4°C under dim red light. The column was equilibrated in and eluted with 25 mM HEPES pH 7.5, 4 mM EDTA, and 1 mM MgCl2, and 1 mL fractions were collected. Each fraction was assayed for A260nm and A280nm , and for radioactivity (100 μL). Aliquots (750 μL) from the protein containing fractions were combined and the UV-vis spectrum recorded. These fractions were then lyophilized to dryness (excluding light). Aliquots (750 μL) from the small molecules fractions (pooled when A260 > A280) were combined and lyophilized to dryness (excluding light). These samples were dissolved in 500 μL water, and the UV-vis spectra recorded. The spectrometer baseline was determined by lyophilizing an equal volume of buffer identical to that used in the experimental samples, which was redissolved in 500 μL of water. The visible spectra of protein-associated cobalamin products were quantified by comparison to a standard of glutathionine cobalamin (GSCbl).(19) The corrin species not associated with the protein were deconvoluted through linear combinations of the spectra of AdoCbl and HOCbl standards in proportions ranging from 1:0 AdoCbl:HOCbl to 0:1 AdoCbl:HOCbl in 0.05 eq. increments. The samples were scaled to match the A525nm of the experimental sample, and subtracted. Characterization of major nucleoside product(s) isolated from a NaBH4 quenched RTPR/ F2CTP inactivation mixture The reaction mixture contained in a final volume of 2 mL: RTPR (125 μM), dATP (500 μM), AdoCbl (125 μM), F2CTP (125 μM), HEPES (25 mM, pH 7.5), EDTA (4 mM), and MgCl2 (1 mM). The inactivation mixture was quenched at 2 min with 500 μL of 250 mM NaBH4 in 500 mM Tris pH 8.5 in a 4.0 mL falcon tube and incubated 5 min at 37°C. The NaBH4 solution was prepared by combining solid NaBH4 with the buffer immediately before use. Vigorous foaming occurred during the inactivation. The solution was then filtered through a YM-30 Lohman et al. Page 2 Biochemistry. Author manuscript; available in PMC 2011 February 23. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript membrane for 15 min at 14,000 × g at 4°C, and the flow through was treated with 200 U alkaline phosphatase for 2 h at 37°C, followed by filtration through a second YM-30 membrane. The sample was acidified by addition of glacial acetic acid and the resulting mixture was lyophilized to dryness to hydrolyze borate esters. The residue was then taken up in 1 mL 10 mM NH4OAc and purified on an Altech Adsorbosphere Nucleotide Nucleoside C-18 column (250 mm × 4.6 mm) using a 2 mL injection loop with elution at a flow rate of 1 mL/min The solvent system for elution was composed of Buffer A (10 mM NH4OAc, pH 6.8) and Buffer B (100 % methanol). The products were eluted with 100 % A for 10 min followed by a linear gradient to 40% B over 25 min and then to 100 % B over 5 min. Under these conditions the standards eluted as follows (compound, retention time): cytosine, 5.7 min; uracil, 7.9 min; cytidine (C), 12.6 min; arabinocytidine (ara-C), 17.4 min; deoxycytidine (dC), 19.0 min; and F2C, 23.2 min. Diode array detection of the eluent allowed identification of cytosine containing nucleosides between 17 min and 22 min. These fractions were pooled and the recovery of cytosinecontaining nucleosides determined to be ~60 nmol based on A270nm. This material was lyophilized to dryness, taken up in 1 mM NH4OAc, and re-purified using the same elution program, substituting 1 mM NH4OAc, pH 6.8, for buffer A and retaining buffer B (100% MeOH). The major cytosine-containing material eluted at 16.2 min and was collected (~35 nmol), lyophilized, and rechromatographed a third time. The final recovery of nucleoside was typically 8–12 nmol. NMR and ESI MS: 1H-NMR (500 MHz, D2O) δ: 7.71 (d, J = 7.5 Hz, 1H, H6), 5.99 (d, J = 6.1 Hz, 1H, H1′), 5.85 (d, J = 7.5 Hz, 1H, H5), 4.41 (dd, J = 5, 6 Hz, 1H, H2′), 4.24 (dd, J = 4.3 Hz, 4.9 Hz, 1H, H3′), 4.02 (m, 1H, H4′), 3.80 (dd, J = 4.0, 12 Hz, 1H, H5′), 3.75 (dd, J = 7.0, 12 Hz, 1H, H5′′). ESI-MS (C9H13N3O5) m/z (M + Na+) calcd 266.0747, obsd 266.0743; (M + H+) calcd 244.0928, obsd 244.0921. Characterization of major nucleoside product isolated from NaBD4 quenched RTPR/F2CTP inactivation mixture A reaction was run identically to that described above, except that NaBD4 was substituted for NaBH4. The final recovery of the trapped nucleotide after three purifications was ~5–8 nmol. 1H-NMR (500 MHz, D2O) δ: 7.71 (d, J = 7.5 Hz, 1H, H6), 5.98 (s, 1H, H1′), 5.84 (d, J = 7.5 Hz, 1H, H5), 4.01 (dd, J = 4.0 Hz, 7.1 Hz, 1H, H4′), 3.80 (dd, J = 4.0, 12 Hz, 1H, H5′), 3.75 (dd, J = 7.0, 12 Hz, 1H, H5′′). High Frequency EPR/ENDOR spectroscopy Samples for D-band (130 GHz) EPR analysis were prepared from a reaction mixture which contained in a final volume of 50 μL: RTPR (300 μM), dATP (1 mM), AdoCbl (450 μM), F2CTP (unlabeled, [1′-2H], or [3′-2H]) (300 μM), TR (20 μM), TRR (0.5 μM), NADPH (1 mM), HEPES (25 mM, pH 7.5). The dATP and reductants were mixed, followed by addition of RTPR. The AdoCbl was then added and mixed in low light, and the inhibition was initiated by the addition of F2CTP or [1′-H]-F2CTP. The reaction mixture was drawn up into the Dband EPR sample tube (O.D. 0.55 mm, I.D. 0.4 mm) mounted in the plastic tip of a pipetteman and then quenched in isopentane cooled with liquid nitrogen. The reaction time was 30 s. The tubes were mounted in the D-band probe under liquid nitrogen and pulsed EPR/ENDOR spectra were obtained on a spectrometer described elsewhere(20,21) using parameters listed in the figure legend to Figure 4. The field for D-band EPR spectra was calibrated using Mn2+ doped in MgO.(22)