Inactivation of lactobacillus leichmannii ribonucleotide reductase by 2',2'-difluoro2'-deoxycytidine s'-triphosphate: Covalent modification

Article abstract of DOI:10.1021/bi902132u

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]F ₂CTP 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.

Full text of DOI:10.1021/bi902132u

1404 Biochemistry 2010, 49, 1404–1417
DOI: 10.1021/bi902132u
Inactivation of Lactobacillus leichmannii Ribonucleotide Reductase by 20,20-Difluoro-
20-deoxycytidine 50-Triphosphate: Covalent Modification†
Gregory J. S. Lohman‡ and JoAnne Stubbe*,‡,§
‡Department of Chemistry and §Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received December 11, 2009; Revised Manuscript Received January 18, 2010
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 20,20-difluoro-20-deoxycytidine 50-tripho-
sphate (F2CTP). [10-3H]- 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 [10-3H]F2CTP and [10-2H]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
[10-3H]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 20-fluoro-20-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.
The nucleoside 2-deoxy-20,20-difluorocytidine (F2C1 or
Gemzar) is a clinically used anticancer drug that targets ribonu-
cleotide reductase (RNR) and DNA polymerase in addition to
several other proteins involved in nucleotide metabolism (1-3).
Studies have shown that F2C is therapeutically effective against a
range of cancers (4-17) with tolerable levels of toxicity. The
mechanism by which F2C inactivates RNRs has been the subject
of a number of investigations (18-21), modeled on our detailed
understanding of the mechanism by which 20-substituted
20-deoxynucleotides inhibit these enzymes (Scheme 1) (22). These
studies revealed that gemcitabine 50-diphosphate (F2CDP) in the
case of the class I RNRs (Escherichia coli and humans forms are
both diphosphate reductases, RDPR) and gemcitabine 5-tripho-
sphate (F2CTP) in the case of the class II RNR from Lactoba-
cillus leichmannii (a nucleoside triphosphate reductase, RTPR)
are potent, mechanism-based inhibitors (20, 21, 23-25).
inhibitors of RNRs, a large number of studies have led to the
general mechanism of inhibition shown in Scheme 1 (X = Cl-,
F-, or N3-). In this mechanism, a protein thiyl radical initiates
the reduction process by abstraction a 30-hydrogen atom from the
nucleotide, which is followed by rapid loss of the 20-substituent.
The glutamate residue in the RNR active site likely facilitates this
process by deprotonation of the 30-hydroxyl, and a cysteine
facilitates C-X bond cleavage by protonation, depending on X
(OH, Cl-, F-, or N3 ). The 30-ketone with an adjacent 20-radical
-
(3, Scheme 1) is produced in all cases. This intermediate then
experiences different fates depending on the conformation on the
nucleotide in the active site, the charge on the leaving group, and
the protonation state of the protein residues. In some cases, 3 is
reduced from the top face (β face of the nucleotide) and in others
from the bottom face (R face). The 30-ketodeoxynucleotide 4 is
generated in both pathways. Recent computational studies have
suggested that when X is anionic, the hydrogen bond network on
the R face of the nucleotide is distinct from the hydrogen bonding
when neutral water is released (26). The binding affinity of 4 for
the enzyme was calculated to be substantially reduced with X-
released relative to H2O. Furthermore, when X- was F-, the
calculations suggested that it was released prior to dissociation of
4. When 4 dissociates from the active site, our previous studies
showed that it decomposes in solution to generate free nucleic
acid base, pyrophosphate (tripolyphosphate), and a reactive
furanone (5). In the case of top face reduction (pathway A),
alkylation is responsible for inactivation. In the case of β face
reduction (pathway B), alkylation and destruction of the radical
initiator are responsible for inactivation.
Since the discovery of Thelander and Eckstein that 20-chloro-
20-deoxynucleotides and 20-azido-20-deoxynucleotides are
†Funding for this study was provided in part by National Institutes of
Health Grant GM-29595.
*To whom correspondence should be addressed. Telephone: (617)
253-1814. Fax: (617) 258-7247. E-mail: stubbe@mit.edu.
1Abbreviations: RNR, ribonucleotide reductase; Gemzar or F2C,
20,20-fluoro-20-deoxycytidine; F2CDP, 20,20-difluoro-20-deoxycytidine
50-diphosphate; F2CTP, 20,20-difluoro-20-deoxycytidine 50-triphosphate;
RTPR, ribonucleoside triphosphate reductase; AdoCbl, adenosylcoba-
lamin; SF, stopped flow; RFQ, rapid freeze quench; SA, specific
activity; dCK, deoxycytidine kinase; TR, thioredoxin; TRR, thioredox-
in reductase; TEAB, triethylammonium bicarbonate; PEP, phosphoe-
nolpyruvate; SEC, size exclusion chromatography; PSD, post-source
decay; HMDS, hexamethyldisilazide; R, RNR large subunit; β, RNR
small subunit.
r
pubs.acs.org/Biochemistry
Published on Web 01/20/2010
2010 American Chemical Society

Article
Biochemistry, Vol. 49, No. 7, 2010 1405
Scheme 1: Proposed Mechanism of Inactivation of RNR by 20-Substituted Mechanism-Based Inhibitors
Studies with gemcitabine differ from those with other
20-substituted mechanism-based inhibitors in that there are two
substituents at C-20. In fact, our initial studies of RNRs from
E. coli and L. leichmannii suggested that the mechanism differed
from the paradigm in Scheme 1 (20, 21). In the case of RTPR, its
incubation with 1 equiv of F2CTP resulted in a 90% loss of
catalytic activity within 30 s. Analysis of products after 30 min
revealed that that the inactivation was accompanied by release of
both fluorides, formation of 0.84 equiv of 50-deoxyadenosine
from the adensoylcobalamin (AdoCbl) cofactor, and ultimately
covalent attachment of cobalamin to C419, one of the cysteines
located on the R face of the nucleotide involved in supplying
the reducing equivalents for the reduction. In addition, kine-
tic studies by stopped flow (SF) spectroscopy revealed that
cob(II)alamin was generated with a kobs of 50 s-1 when 1 equiv
of F2CTP was reacted with RTPR, suggesting that the rate
constant would be similar to that of cob(II)alamin formation
with CTP (250 s-1), if the nucleotide was present at saturating
levels. Rapid freeze quench (RFQ) EPR experiments showed
initial formation of the same exchange-coupled thiyl radical-
cob(II)alamin species (22 ms) observed with CTP, followed by
stoichiometric conversion to a new radical-cob(II)alamin spe-
cies over 255 ms, suggesting thiyl radical-mediated nucleotide
radical formation. Efforts to further study the mechanism of this
inactivation were hampered by the unavailability of the isotopi-
cally labeled gemcitabine analogues.
was critical for identification by mass spectrometric methods, the
peptides modified by a sugar moiety derived from F2CTP. In the
preceding paper (DOI: 10.1021/bi9021318), [10-2H]F2CTP has
been used to establish that the new radical generated by the thiyl
radical-cob(II)alamin species is nucleotide-derived (27). We also
describe the fate of the adenosylcobalamin and the structure of an
unusual nucleotide trapped with NaBH4 which we believe is
derived from the observed nucleotide radical intermediate. These
studies together have allowed us to formulate mechanisms for
RTPR inactivation that, while complex, do follow the paradigms
shown in Scheme 1.
MATERIALS AND METHODS
2-Deoxy-3,5-di-O-benzoyl-2,2-difluororibonolactone was
a
gift of Eli Lilly & Co. All reagents were purchased from Sigma
or Mallinckrodt and used without further purification unless
otherwise indicated. Analytical TLC was performed on an
E. Merck silica gel 60 F254 plate (e0.25 mm). Compounds were
visualized by cerium sulfate-ammonium molybdate stain and
heating or by exposure to UV light. Human deoxycytidine kinase
(dCK) expression plasmids were a gift from S. Eriksson (28), and
the protein [specific activity (SA) of 150 nmol mg-1 min-1] was
expressed, purified, and stored in 20 mM Tris (pH 7.9), 0.5 M
NaCl, 10 mM DTT, and 20% glycerol. UMP/CMP kinase
expression plasmids were a kind gift from A. Karlsson (29).
The protein was expressed and purified (SA of 2.1-4.8 μmol
mg-1 min-1) as described previously (30) and stored in 50 mM
Tris (pH 8.0) and 10 mM reduced glutathione. Its concentration
was determined by a Bradford assay with a BSA standard.
Inorganic pyrophosphatase (baker’s yeast) (SA of 868 units/
mg) was obtained from Sigma as a lyophilized powder and taken
up in 165 mM Tris (pH 7.2) immediately prior to use. Pyruvate
kinase (rabbit muscle) (SA of 450 units/mg) was obtained from
Sigma as a lyophilized powder and prepared as a 1200 units/mL
We now report synthesis of 10-2H-, 10-3H-, and 5-3H-labeled
gemcitabine nucleotides by a combination of chemical and
enzymatic methods. Using these probes, we now report, in this
paper and the preceding paper (DOI: 10.1021/bi9021318), on the
mechanism by which F2CTP inhibits the class II L. leichmannii
RTPR (27). We demonstrate that 0.47 equiv of sugar from
F2CTP covalently labels RTPR and that in contrast to our
previous studies, cytosine (∼0.7 equiv) is released to solution. Use
of a mixture of [10-2H,3H]F2CTP for the inactivation of RTPR

1406 Biochemistry, Vol. 49, No. 7, 2010
Lohman and Stubbe
stock in 50 mM Tris (pH 7.5), 80 mM KCl, and 20 mM MgCl2.
Calf intestine alkaline phosphatase was purchased from Roche as
a 20 units/μL stock in 100 mM Tris (pH 8.5). E. coli thioredoxin
(TR, SA of 40 units/mg) and E. coli thioredoxin reductase (TRR,
SA of 1320 units/mg) were isolated as previously described
(31, 32). RTPR (SA of 0.38-0.42 μmol mg-1 min-1) was isolated
as previously described (33), omitting the second anion exchange
column. AdoCbl was handled with minimal exposure to light. All
reaction mixtures including AdoCbl were kept wrapped in foil at
all times. Transfer of AdoCbl-containing solutions was per-
formed under dim or red light conditions. The following extinc-
tion coefficients were used: λ280 = 56755 at pH 7.0 for dCK,
λ268 = 9360 at pH 7.0 for F2C (34), λ271 = 9100 at pH 7.0 for
cytidine, λ267 = 6100 at pH 7.0 for cytosine, and λ259 = 15400 at
pH 7.0 for adenosine (35). UV-vis spectra were recorded on a
Cary-3 spectrometer. Anion exchange column fractions were
assayed using an Ultramark Bio-RAD fixed wavelength plate
reader. Scintillation counting was performed using Emulsifier-
Safe liquid scintillation counting cocktail (Perkin-Elmer) on a
Beckman LS6500 multipurpose scintillation counter.
Water (1 mL) was then added slowly. Foaming occurred as H2
gas evolved. The flask was equipped with a reflux condenser, and
the mixture was refluxed for 30 min and then cooled to 0 °C and
the reflux condenser removed. Hydrogen peroxide (1 mL, 30%
solution) was added dropwise over 5 min, along with enough 2 N
NaOH to keep the pH at 7.5-8.0 (monitored with a microelec-
trode, ∼200 mL over the course of the quench). The mixture was
poured into EtOAc (100 mL) and washed with water (2 ꢀ 50
mL). The organic layer was dried over MgSO4 and filtered and
the solvent removed in vacuo.
Residue 7 (Scheme S1 of the Supporting Information) was
dried over P2O5 and dissolved in CH2Cl2 (5 mL). The reaction
mixture was cooled to 0 °C, and triethylamine (TEA, 0.5 mL,
distilled from KOH) and mesyl chloride (0.39 mL, 5 mmol) were
added. The ice bath was removed and the reaction mixture stirred
for 3 h at room temperature. The reaction mixture was diluted to
100 mL with CH2Cl2 and the organic layer washed with 1 N HCl,
water, saturated NaHCO3, and water. The organic layer was
dried over MgSO4 and filtered and the solvent removed in vacuo.
Purification using silica gel chromatography (1 cm ꢀ 15 cm
column, 4:1 hexane/ethyl acetate elutant) yielded 8 (143 mg, 0.313
mmol, 63%) as a clear oil. The 1H NMR spectrum was consistent
with previously reported data (34).
Synthesis of [10-3H]-2-Deoxy-3,5-di-O-benzoyl-2,2-di-
fluoro-1-O-methanesulfonyl-D-ribofuranoside (8, Scheme
S1 of the Supporting Information) (36, 37) and [10-3H]-
F2C. F2C was synthesized from 2-deoxy-3,5-di-O-benzoyl-2,2-
difluororibonolactone (6) or the unblocked lactone by modifica-
tions of published procedures, and the reaction is summarized in
Scheme S1 of the Supporting Information (34). Only the step to
incorporate the tritium label at C-10 is described in detail. All
solid reagents were dried over P2O5 under vacuum overnight. All
solvents and liquid reagents were distilled from CaH immediately
before use, except mesyl chloride, which was distilled from P2O5.
NaB3H4 (500 mCi of NaB3H4, SA of 14.22 Ci/mM, Perkin-
Elmer) was combined with 152 mg or 4 mmol of NaBH4 and the
mixture dissolved in diglyme (4 mL) under N2 to make a 1.0 M
solution. A three-neck flask fitted with a coldfinger, gas inlet, and
dropping funnel was assembled and connected to a 10 mL conical
flask through an exit from the coldfinger using tygon tubing and
a needle. This apparatus allowed B2H6 generated in the three-
neck flask to be transferred to the 10 mL flask. The entire
apparatus was flame-dried and cooled under N2. The coldfinger
attached to the three-neck flask was filled with dry ice and
acetone. The NaB3H4 solution was added to the dropping funnel,
and boron trifluoride diethyl etherate (1.25 mL, 10 mmol,
2.5 equiv) was added to the three-neck flask in an oil bath at
room temperature. The conical flask was immersed in a dry ice/
acetone bath and tetrahydrofuran (THF, 2 mL) added. The
needle was immersed in the THF and the N2 flow turned to a
minimum. The borohydride solution was added dropwise over
1 h. The evolution of gas was immediately evident, and a white
precipitate (NaBF4) (38) formed over the course of the reaction.
The oil bath was heated to 70 °C for 30 min to distill off any
remaining B2H6. The apparatus was disassembled, leaving the
conical flask under N2 in the dry ice/acetone bath. The concen-
trations of BH3 and THF were established by titration (36, 37).
The BH3/THF solution was transferred to an ice/salt bath
(-15 °C), and 2-methyl-2-butene (1.03 mL, 9.6 mmol) was added
slowly, dropwise via syringe over 1 h. The reaction mixture was
stirred for an additional 1 h at -15 °C. The solution of
disiamylborane was transferred to an ice bath (0 °C), and a
solution of 6 (188 mg, 0.5 mmol) in 1 mL of THF was added
dropwise via syringe over 30 min. The reaction mixture was
stirred for 1 h at 0 °C followed by 16 h at room temperature.
Bis(trimethylsilyl)cytosine (9) was made from cytosine (39)
and coupled to 8 using trimethylsilyl trifluoromethanesulfonate
as previously described (34). Compound 10 as a 6:4 R/β mixture
was obtained and quantified by UV absorbance (ε268 = 9360 at
pH 7.0) to give 190.5 mmol (76% from 8). 1H NMR was
consistent with that reported for 10 (34), and it was judged to
be >95% pure.
20-Deoxy-20,20-difluorocytidine 50-Monophosphate
(F2CMP) (28, 30). F2C was converted to its monophosphate
directly from the R/β mixture. The reaction mixture contained, in
a final volume of 1 mL, 5 mM β-F2C, 10 mM ATP, 2 mM DTT,
0.5 mg/mL BSA, 1.4 mg/mL human dCK, 50 mM Tris (pH 7.6),
100 mM KCl, and 10 mM MgCl2. The reaction was initiated by
addition of dCK and the mixture incubated at 37 °C for 45 min.
The reaction mixture was loaded on a DEAE-Sephadex A-25
column (20 mL, 20 cm ꢀ 1 cm) equilibrated in 5 mM triethyl-
ammonium bicarbonate (TEAB) (pH 6.8) and the column
washed with 50 mL of 5 mM TEAB. The product was eluted
using a 100 mL ꢀ 100 mL linear gradient from 5 to 400 mM
TEAB. Fractions (5 mL) were assayed for A260; the nucleotide-
containing fractions were combined, and the solvent was re-
moved in vacuo. 1H NMR analysis of the flow-through from the
initial wash showed only the presence of the R anomer of 10 (34).
F2CMP eluted at 250 mM TEAB (4.45 mmol, 90%): 1H NMR
(500 MHz, D2O, selected peaks) δ 7.83 (d, J = 7.5 Hz, 1H), 6.15
(t, J = 6.4 Hz, 1H), 6.05 (d, J = 7.5 Hz, 1H), 4.36 (dd, J = 12.4,
21.1 Hz, 1H), 4.02-4.09 (m, 2H), 3.91-3.96 (m, 1H); 31P NMR
(121.5 MHz, D2O, phosphoric acid external reference) δ 4.6.
20-Deoxy-20,20-difluorocytidine 50-Diphosphate (F2CDP)
(29, 30, 40). The reaction mixture contained, in a final volume
of 5 mL, 2 mM F2CMP, 8 mM ATP, 2 mM DTT, 50 mM Tris
(pH 8), 25 mM MgCl2, and 66.5 mg/mL UMP-CMP kinase. The
reaction mixture was incubated for 30 min at 37 °C, diluted to
50 mL with cold water, and loaded onto a DEAE-Sephadex A-25
column (30 mL, 18 cm ꢀ 1.5 cm). The column was washed with
water (100 mL) and the product eluted with a 400 mL ꢀ 400 mL
linear gradient from 0 to 600 mM TEAB. The fractions (7.5 mL)
were assayed by A260, with F2CDP eluting along with ADP at
450 mM TEAB. The fractions were combined, and the solvent

Article
Biochemistry, Vol. 49, No. 7, 2010 1407
was removed in vacuo. The product was coevaporated with 50%
ethanol in water (5 ꢀ 20 mL) to remove excess TEAB. To remove
contaminating adenosine nucleotides, this material was dissolved
in water (24 mL) and NaIO4 (0.5 mmol, 1 mL of a 0.5 M solution)
was added. The reaction mixture was incubated for 10 min at
37 °C, and then methylamine (2.5 mmol, 640 mL of a 3.9 M
solution in water, pH adjusted to 7.5 with phosphoric acid) was
added and the reaction mixture incubated for an additional 20
min. The reaction was quenched by addition of rhamnose
(1 mmol, 1 mL of a 1 M solution). To this mixture was added
6.6 mL of 8 mM MgCl2, 165 mM Tris (pH 7.2), and yeast
inorganic pyrophosphatase [165 mL of a 50 units/mL stock in
165 mM Tris (pH 7.2)] to give final concentrations of 2 mM
MgCl2, 55 mM Tris (pH 7.2), and 0.25 units/mL inorganic
pyrophosphatase. This mixture was incubated for 45 min at
37 °C, diluted to 300 mL with cold water, and loaded onto a
DEAE-Sephadex A-25 column (30 mL, 18 cm ꢀ 1.5 cm). The
column was washed with water (100 mL) and the product eluted
with a 400 mL ꢀ 400 mL linear gradient from 0 to 600 mM
TEAB. The fractions (7.5 mL) were assayed by A260; the F2CDP-
containing fractions eluting at 450 mM TEAB were combined,
and the solvent was removed in vacuo. The product was
RTPR Assay (spectrophotometric). The assay mixture
contained, in a final volume of 500 μL, RTPR (typically 0.25,
0.5, or 1 μM), CTP (1 mM), dATP (100 μM), NADPH (0.2 mM),
TR (20 μM), TRR (0.2 μM), HEPES (25 mM, pH 7.5), EDTA
(4 mM), and MgCl2 (1 mM). The reaction was initiated with
AdoCbl (20 μM) at 37 °C and monitored by the change in A340
(ε = 6220 M-1 cm-1). The SA of RTPR was typically 0.28-0.44
mmol mg-1 min-1
.
RTPR Assay for Monitoring [14C]dCTP. The assay
mixture contained, in a final volume of 300 μL, RTPR (1 μM),
[14C]CTP (SA of 1500-2500 cpm/nmol, 1 mM), dATP (100 μM),
NADPH (1 mM), TR (20 μM), TRR (0.2 μM), HEPES (25 mM,
pH 7.5), EDTA (4 mM), and MgCl2 (1 mM). The reaction was
initiated with AdoCbl (20 μM) and the mixture incubated at
37 °C; a control aliquot was removed before addition of AdoCbl.
Four aliquots (50 μL) were removed at time points over 5 min,
and the reaction was quenched by addition of 2% perchloric acid
(25 μL). The aliquots were neutralized with 0.4 M KOH (25 μL)
after all time points were collected. Cytosine (50 nmol) and dC
(50 nmol) were added to each aliquot; 20 units of alkaline
phosphatase in 500 mM Tris (pH 8.6) and 1 mM EDTA
(26.5 μL) was added, and the aliquots were incubated for 2 h
at 37 °C. The dCDP was quantitated by the method of Steeper
and Steuart (41).
Time-Dependent Inactivation of RTPR with F2CTP
(19, 21). The inactivation mixture contained, in a final volume
of 100 μL, prereduced RTPR (5 μM), dATP (100 μM), NADPH
(1 mM), TR (20 μM), TRR (0.2 μM), AdoCbl (20 μM), HEPES
(25 mM, pH 7.5), EDTA (4 mM), and MgCl2 (1 mM). To study
inactivation in the absence of reductant, NADPH, TR, and TRR
were omitted. The inactivation was initiated by addition of
F2CTP (or isotopically labeled derivative, 5 μM) and the reaction
conducted at 37 °C. The assay for activity was conducted by
measuring [14C]dCTP production.
Size Exclusion Chromatography (SEC) on RTPR In-
activated with [10-3H] or [5-3H]F2CTP. Inactivation mixtures
were prepared as described above in a final volume of 500 μL.
Experiments were conducted both in the presence and in the
absence of reductants. Inactivations were initiated by addition of
F2CTP (10-3H-labeled, SA of 7200 cpm/nmol; or 5-3H-labeled,
SA of 10800 cpm/nmol) and incubated for 2 min at 37 °C. An
aliquot was assayed for activity using the spectrophotometric
assay before initiation and at the inactivation end point. After
2 min, an aliquot (200 μL) was loaded on Sephadex G-50
columns (1 cm ꢀ 20 cm, 20 mL), equilibrated with and eluting
with 25 mM HEPES (pH 7.5), 1 mM MgCl2, and 4 mM EDTA,
and 1 mL fractions were collected. A second aliquot (210 μL) was
mixed with 630 μL of 8 M guanidine and then loaded on a
Sephadex G-50 column (1 cm ꢀ 20 cm, 20 mL) equilibrated in and
eluted with 2 M guanidine in assay buffer, and 800 μL fractions
were collected. In all cases, fractions were assayed for A260 and A280
and for radioactivity by scintillation counting (500 μL aliquots).
The recovery of radioactivity was typically >95%. The recovery of
protein determined from A280 was typically >90%.
1
redissolved in water (1 mL) giving 5.6 mmol (56% yield): H
NMR (500 MHz, D2O) δ 7.71 (d, J = 7.7 Hz, 1H), 6.09 (t, J =
7.2 Hz, 1H), 5.96 (d, J = 7.7 Hz, 1H), 4.40 (dd, J = 13.3, 22.2 Hz,
1H), 4.05-4.21 (m, 2H), 3.99-4.03 (m, 1H); 31P NMR (121.5
MHz, D2O, phosphoric acid external reference) δ -9.5 (d, J =
22.1 Hz), -10.6 (d, J = 22.1 Hz). The 31P NMR showed no
contamination with inorganic pyrophosphate.
20-Deoxy-20,20-difluorocytidine 50-Triphosphate (F2CTP).
The reaction mixture (20 mL) contained 2 mM F2CDP, 4 mM
phosphoenolpyruvate (PEP), 50 mM Tris (pH 7.5), 80 mM KCl,
20 mM MgCl2, and 120 units/mL pyruvate kinase. The reaction
mixture was incubated at 37 °C for 1 h. The mixture was then
diluted with cold water (50 mL) and loaded on a DEAE-
Sephadex A-25 column (60 mL, 20 cm ꢀ 2 cm), and the column
was washed with water (100 mL) and the product eluted with a
400 mL ꢀ 400 mL linear gradient from 0 to 750 mM TEAB. The
fractions (10 mL) were assayed for A260. The triphosphate-
containing fractions eluting at 550 mM TEAB were combined,
and the solvent was removed in vacuo. The product was dissolved
in 5 mL of ddH2O to give 35 mmol (88% yield): 1H NMR (500
MHz, D2O, selected peaks) δ 7.75 (d, J = 7.7 Hz, 1H), 6.09
(t, J = 7.2 Hz, 1H), 6.02 (d, J = 7.3 Hz, 1H), 4.33-4.40 (m, 1H),
4.21-4.26 (m, 1H), 4.10-4.14 (m, 1H), 4.03-4.06 (m, 1H); 31P
NMR (121.5 MHz, D2O, phosphoric acid external reference)
δ -9.3, -10.5, -21.4.
[10-2H]F2C. The synthesis of 8 was repeated with the sub-
stitution of NaB2H4 (98% 2H, Sigma) for NaBH4. The yield of
[10-2H]F2C (274 mmol) was 64%. The level of incorporation of
2H was 93% as determined by 1H NMR and ESI-MS. The
corresponding mono-, di-, and tri-phosphates were prepared
enzymatically in 86, 60, and 76% yield, respectively.
[10-3H]F2CTP. Enzymatic phosphorylations to the mono-,
di-, and triphosphates were conducted as described above to give
85% (SA of 8700 cpm/nmol), 60% (SA of 8620 cpm/nmol), and
88% (SA of 8620 cpm/nmol), respectively.
Elimination of Cytosine Deaminase Activity from Pre-
reduced RTPR. An RTPR stock solution (∼1 mM, 250 μL) was
mixed with 5 mM o-phenanthroline (750 μL), and DTT was
added to a final concentration of 30 mM. The solution was
incubated for 20 min at 37 °C, and RTPR was isolated by
Sephadex G-25 chromatography and quantified by standard
procedures. Alternatively, the cytosine deaminase was removed
by SEC on a Sephacryl S-300 column.
[5-3H]F2CTP. [5-3H]F2CDP was synthesized from [5-3H]-
F2C provided by Eli Lilly (30). [5-3H]F2CDP (2.2 μmol) was
converted to the triphosphate by the PEP/pyruvate kinase
procedure described above. The yield was 1.85 mmol (85%) with
a SA of 10060 cpm/nmol.

1408 Biochemistry, Vol. 49, No. 7, 2010
Lohman and Stubbe
Spectrophotometric Detection of Cytosine Deaminase
Activity in the RTPR Preparation. The conversion of cyto-
sine (λmax = 269 nm) to uracil (λmax = 258 nm) was monitored
spectrophotometrically. The reaction mixture contained, in a
volume of 500 μL, 50 μM cytosine, 5 μM RTPR, 20 μM AdoCbl,
5 mM DTT, 25 mM HEPES (pH 7.5), 1 mM MgCl2, and 4 mM
EDTA. Three different preparations of RTPR were assayed: as
isolated, purified by S-300 SEC, or pretreated with 3 mM
o-phenanthroline. The UV spectra were recorded after 0 min,
30 min, 1 h, 6 h, and 1 day.
Analysis of Cytosine Generated during the Inactivation
of RTPR with F2CTP. The reaction mixture contained, in a
final volume of 220 μL (or 1020 μL), prereduced RTPR (50 μM),
dATP (500 μM), AdoCbl (50 μM), HEPES (25 mM, pH 7.5),
EDTA (4 mM), and MgCl2 (1 mM). [5-3H]F2CTP (50 μM, 7200
cpm/nmol) or [5-3H]F2CTP (50 μM, 10800 cpm/nmol) was added
to initiate the reaction, and the mixture was then incubated for
2 min at 37 °C. Before initiation with F2CTP and after the
inactivation was complete, 10 μL aliquots were removed and
assayed for RTPR activity by the spectrophotometric assay. At
2 min, the inactivation mixture was filtered through a YM-30
membrane (30000 molecular weight cutoff minicon) for 10 min at
14000g and 4 °C. F2C (50 nmol) and cytosine (50 nmol) were
added as carriers before filtration. Twenty units of alkaline
phosphatase was added to the filtrate, and the reaction mixture
was incubated for 3 h at 37 °C and filtered again using a YM-30
centricon.
The sample was then purified by HPLC using a Rainin SD-200
HPLC system and an Altech Adsorbosphere Nucleotide Nucleo-
side C-18 column (250 mm ꢀ 4.6 mm) with elution at a flow rate
of 1 mL/min. The solvent system consisted of buffer A [10 mM
NH4OAc (pH 6.8)] and buffer B (100% methanol). The flow
program was 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, standards eluted as follows: cytosine at 5.7 min, uracil
at 7.9 min, C at 12.6 min, ara-C at 17.4 min, dC at 19.0 min, and
F2C at 23.2 min. Fractions (1 mL) were collected; for samples
with radiolabeled F2CTP, 200 μL of each fraction was analyzed
by scintillation counting. The cytosine peak was isolated and
recovery calculated on the basis of the carrier added and
scintillation.
volume of 220 μL, prereduced RTPR (50 μM), dATP (500
μM), AdoCbl (75 μM), HEPES (25 mM, pH 7.5), EDTA
(4 mM), and MgCl2 (1 mM). [10-3H]F2CTP (50 μM, SA of
7200 cpm/nmol) was added to initiate the reaction, and the
reaction mixture was incubated for 2 min at 37 °C. Before
initiation with F2CTP and after the inactivation was complete,
10 μL aliquots were removed and assayed for RTPR activity. At
2 min, the inactivation mixture was quenched with 50 μL of 250
mM NaBH4 and 400 mM Tris (pH 8.3) and incubated for an
additional 5 min at 37 °C. The NaBH4 solution was prepared via
combination of solid NaBH4 with the buffer solution immedi-
ately before use; vigorous foaming occurred upon addition to the
inactivation mixture. To this mixture was added 750 μL of 8 M
guanidine, 40 mM DTT, 5.33 mM EDTA, and 400 mM Tris (pH
8.3), and the reaction mixture was incubated for 30 min at 37 °C.
Iodoacetamide was added to a final concentration of 250 mM
(25000 equiv per RTPR) and the reaction mixture incubated for
an additional 1 h at 37 °C. The protein was then separated from
small molecules on a Sephadex G-50 column (1 cm ꢀ 20 cm,
20 mL) equilibrated into 0.1 M NH4HCO3 (pH 8.2) [or 0.1 M
NH4HCO3 (pH 8.2) and 2 M urea]. The protein-containing
fractions were combined (typically 2 mL total volume, 90%
recovery of protein, >95% recovery of loaded radioactivity, 0.28
equiv of radioactivity coeluted with protein). Trypsin
(Worthington) was added from a freshly prepared stock solution
to a final RTPR:trypsin concentration ratio of 4:1 (w/w) and
incubated for 2 h at 37 °C. The reaction was quenched by
addition of 25 μL of trifluoroacetic acid (TFA, to pH ∼2), and
the peptides were immediately separated using a Waters 2487
HPLC system with a Phenomenex Jupiter C18 peptide column
˚
(150 mm ꢀ 4.6 mm, 5 μm, 300 A pore size) at a flow rate of 1 mL/
min. The peptides were eluted with buffer A (0.1% TFA in
ddH2O) and buffer B (0.1% TFA in acetonitrile) by a linear
gradient from 0 to 45% B over 90 min. Fractions were collected
(1 mL), and aliquots (100 μL) of each fraction were assayed for
radioactivity by scintillation counting. The recovery of injected
radioactivity was typically 80%. Four regions of radioactivity
were observed (Figure 1). Fractions were pooled on the basis of
the observed peaks of radioactivity, concentrated to <1 mL on a
lyophilizer, and repurified using the same column and flow rate
with buffer A [10 mM NH4OAc (pH 6.8)] and buffer B (100%
acetonitrile) using a gradient from 0 to 35% B over 90 min.
Fractions (1 mL) were collected, and aliquots of each fraction
(250 μL) were assayed for radioactivity. The major peaks of
radioactivity were pooled and submitted for mass spectrometry
analysis without concentration. Reactions were also conducted
as described above substituting NaB2H4 for NaBH4 or F2CTP
that was a mixture of 10-2H- and 10-3H-labeled forms (SA of 7200
cpm/nmol) to give a stock with 60% 10-2H with a SA of 2600
cpm/nmol.
Stability of [3H]RTPR Generated by Inactivation with
[10-3H]F2CTP to Trypsin Digestion Conditions. The mixture
contained, in a final volume of 300 μL, prereduced RTPR
(15 μM), dATP (100 μM), AdoCbl (22.5 μM), HEPES
(25 mM, pH 7.5), EDTA (4 mM), and MgCl2 (1 mM). The
reaction was initiated by addition of [10-3H]F2CTP (SA 7200
cpm/nmol) to a final concentration of 15 μM. The reaction
mixture was incubated for 2 min at 37 °C. The mixture, a 200 μL
aliquot, was passed through Sephadex G-50 columns (1 cm ꢀ
20 cm, 20 mL) equilibrated and eluted in 0.1 M NH4HCO3 (pH 8)
and 2 M urea (trypsin digestion buffer), and the protein-
containing fractions were assayed for A280 and radioactivity.
The sample (1.5 mL) was loaded into a Slidalizer dialysis cassette
(10000 molecular weight cutoff) and dialyzed against the same
buffer in which the sample was eluted. Aliquots (250 μL) were
removed at 1, 2, 4, and 6 h. The dialysis buffers were changed, and
the samples were dialyzed for an additional 14 h. A final aliquot
was removed, and all aliquots were assayed for radioactivity by
scintillation counting.
Peptide Mass Spectrometry (42). MALDI-TOF and
tandem MS/MS mass spectrometry were performed by J. Leszyk
at UMass Medical School on a Kratos Axima CFR (Shimadzu
Instruments) matrix-assisted laser desorption ionization
(MALDI) mass spectrometer. Samples (0.5 mL, containing
50-100 fmol of F2CTP-labeled peptide, determined by specific
activity) were applied to the target and mixed with 0.5 μL of
matrix which was 2,5-dihydroxybenzoic acid at 15 mg/mL in a
50:50 CH3CN/0.1%TFA mixture. Samples were allowed to air-
dry prior to being inserted into the mass spectrometer. Peptides
were analyzed in positive ion mode in midmass range (100-3000
Da) with an accuracy of (100 ppm. The instrument was
Trypsin Digestion of RTPR Inactivated with
[10-3H]F2CTP. The reaction mixture contained, in a final

Article
Biochemistry, Vol. 49, No. 7, 2010 1409
band intensities measured. The band intensities were quantified
using Bio-Rad Quantity One.
SEC on C119S RTPR Inactivated with [10-3H]F2CTP.
SEC experiments were performed with C119S-RTPR, which was
analyzed as described above for wild-type RTPR in the absence
of reductants.
Characterization of Major Nucleoside Product(s) Iso-
lated from a NaBH4-Quenched C119S RTPR/F2CTP In-
activation Mixture. The reaction mixture contained, in a final
volume of 200 μL, C119S-RTPR (50 μM), dATP (250 μM),
AdoCbl (75 μM), F2CTP (50 μM), HEPES (25 mM, pH 7.5),
EDTA (4 mM), and MgCl2 (1 mM). The inactivation reaction
was quenched at 2 min with 50 μL of 250 mM NaBH4 in 500 mM
Tris (pH 8.5) in a 4.0 mL Falcon tube and the mixture incubated
for 5 min at 37 °C. The NaBH4 solution was prepared by using
solid NaBH4 as described above. The solution was then filtered
through a YM-30 membrane for 15 min at 14000g and 4 °C, and
the flow-through was treated with 200 units of alkaline phos-
phatase for 3 h at 37 °C, followed by filtration through a second
YM-30 membrane. The sample was acidified by addition of
glacial acetic acid (5% of the final volume), and the resulting
mixture was lyophilized to dryness to hydrolyze borate
esters. The residue was then taken up in 1 mL of 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 and analysis of nucleosides was described
above for cytosine identification. Fractions were collected
(1 mL), and aliquots (100 μL) of each fraction were assayed
for radioactivity by scintillation counting. The major peak of
radioactivity (43% of total counts) was found to co-elute with a
standard of 20-fluoro-20-deoxycytidine. The presence of this
compound was confirmed: ESI-MS (C9H12FN3O4) m/z (M þ
Naþ) calcd 268.0710, observed 268.0718; (M þ Hþ) calcd
246.0890, observed 246.0901.
F
IGURE 1: HPLC purification of peptides from RTPR inacti-
vated with [10-3H]F2CTP, treated with NaBH4 at 2 min, alkylated
with iodoacetamide, and digested with trypsin. A214 (;) and
counts per minute (---). (A) Initial purification with a linear gradient
(
) with 0.1% TFA from 0 to 45% CH3CN over 90 min: region
3 3 3
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 with a linear gradient ( ) with 10 mM NH4OAc (pH
6.8) from 0 to 35% CH3CN over 90 min (80% recovery of radi-
olabel).
3 3 3
externally calibrated with bradykinin (757.40 Da), P14R (MS
mass standard, Sigma-Aldrich, 1533.86 Da), and adrenocortico-
tropic hormone fragment 18-39 (Sigma-Aldrich, 2465.20 Da).
Post-source decay (PSD) analysis was performed on the same
instrument using a timed ion gate for precursor selection with a
laser power ∼20% higher than that for MS acquisition. PSD
fragments were separated in a curved field reflectron that allowed
for a seamless full mass range acquisition of the spectrum, with an
accuracy of (1000 ppm. All spectra were processed with Mascot
Distiller (Matrix Sciences, Ltd.) prior to database searching (43).
Database searches were performed with Mascot (Matrix
Sciences, Ltd.). For MS searches, the Peptide Mass Fingerprint
program was used with a peptide mass tolerance of 150 ppm. For
MS/MS searching, the MS/MS Ion Search program was used
with a precursor tolerance of 150 ppm and a fragment tolerance
of 1 Da.
SDS-PAGE of RTPR Inactivations with F2CTP. In-
activations were performed as described above. Each sample
contained, in a final volume of 300 μL, prereduced RTPR
(25 μM), dATP (500 μM), AdoCbl (37.5 μM), HEPES
(25 mM, pH 7.5), EDTA (4 mM), and MgCl2 (1 mM). The
reaction was initiated by addition of F2CTP to a final concentra-
tion of 25 μM. Reaction mixtures containing reductant used
5 mM DTT. Aliquots were quenched when mixed with an equal
volume of 2ꢀ Laemlli buffer. An aliquot was removed immedi-
ately after F2CTP addition (0 min), and two aliquots were
removed at 5 min: one was not boiled, and the other was boiled
for 3 min. Samples were analyzed on a 10% SDS-PAGE gel and
RESULTS
Synthesis of Isotopically Labeled F2CTP. The synthesis of
[10-3H]F2C (10) was conducted using modifications of the pre-
viously published procedure and is summarized in Scheme S1 of
the Supporting Information (34). The reduction of the difluori-
nated lactone 6 used disiamylborane made from NaB3H4 and 2
methyl-2-butene. The presence of the two fluorines required an
increased ratio of disiamylborane to 6 relative to ribonolactone to
produce high yields (36). The reduced 7 was then converted
directly to the mesylate 8 with mesyl chloride and TEA without
purification in 85% yield from the lactone 6. The mesylate was
coupled to bis-trimethylsilylcytosine 9 prepared by refluxing
cytosine in a 10:1 HMDS/TMSCl mixture. Efforts to make 9
from cytosine in HMDS using ammonium sulfate as a catalyst
were unsuccessful on the small scale used to make [10-3H]F2C. 9
added to 8 in xylenes and refluxed in the presence of trimethylsilyl
trifluoromethanesulfonate resulted in production of protected
nucleoside as a 6:4 R/β mixture that was directly deprotected with
sodium methoxide to give R/β 10 in 74% yield from 8. The
published protocols separate the F2C anomers by crystallization
at either the benzoate-protected or fully deprotected stage. This
approach is incompatible with the scale used for radiolabeled
syntheses. After investigating a number of options, we deter-
mined that it is most convenient to resolve the R/β mixture
enzymatically during the conversion of F2C to F2CMP.

1410 Biochemistry, Vol. 49, No. 7, 2010
Lohman and Stubbe
Phosphorylation of F2C. F2C is very difficult to phosphor-
ylate chemically using activated phosphates due to the enhanced
acidity of its 30-OH which results in rapid formation of the 30,50-
cyclic phosphate. Previous studies had reported the ability to
generate F2CMP using human dCK and to generate F2CDP
using F2CMP and UMP/CMP kinase (6, 42, 43, 47-49). Treat-
ment of R/β-F2C with human dCK and ATP allowed selective
conversion of the β anomer to the 50-monophosphate (F2CMP)
in 85% yield. The desired product readily separated from the
remaining nucleoside during anion exchange chromatography.
To make the F2CTP, we first made the F2CDP, which also has
been used to investigate the class I RNRs which use NDP
substrates. F2CDP was generated enzymatically from F2CMP
and ATP with human UMP/CMP kinase. However, this proce-
dure yields an equilibrium mixture of starting material and
products: F2CDP and ADP. Since these products co-elute on
anion exchange chromatography, a periodate cleavage step was
introduced to destroy ADP (30, 40). NaIO4 (aqueous) selectively
reacts with the syn-diol of the ADP to cleave the 20-C-30-C bond
generating a dialdehyde. The F2CDP is unreactive. After removal
of excess periodate, MeNH2 (pH 7.5) is added to catalyze
elimination of pyrophosphate from the dialdehyde. Addition of
inorganic pyrophosphatase converts the inorganic pyropho-
sphate to inorganic phosphate. Anion exchange chromatography
of this reaction mixture allows recovery of homogeneous F2CDP
in 56% yield.
In the development of the UMP/CMP kinase procedure for
production of F2CDP, experiments were initially conducted in
the presence of pyruvate kinase and PEP to recycle the ADP to
ATP and prevent the problem of its separation from F2CDP (30).
However, HPLC analysis indicated that the F2CDP was phos-
phorylated under the conditions investigated. This observation
suggested that pyruvate kinase could be used to make F2CTP.
Two equivalents of PEP and 120 units/mL pyruvate kinase
resulted in 88% conversion to F2CTP in 1 h. The enzymatic
phosphorylation methods have been used to prepare all of the
isotopically labeled gemcitabine nucleotides [[5-3H]F2CD(T)P
and [10-2H]F2CD(T)P] in 10-100 μmol quantities.
any cytosine subsequent to the inactivation (21). Given our
results with [5-3H]F2CTP and the observation that cytosine is
released when the class I RDPRs from E. coli or human are
inactivated with F2CDP (20, 23-25), our efforts focused on
determining if cytosine was being converted to uracil. The
inactivation was conducted with [5-3H]F2CTP, and the small
molecules were analyzed by RP-HPLC subsequent to depho-
sphorylation of the nucleotides with alkaline phosphatase. The
3
retention time of the new H-labeled material was identical to
that of a uracil standard. Isolation of this material and examina-
tion by UV and NMR spectroscopy confirmed its identity. These
results suggested that RTPR preparations were contaminated
with E. coli cytosine deaminase (44). Several different types of
experiments confirmed this to be the case and allowed its removal
(see Materials and Methods). Inactivation studies with
[5-3H]F2CTP were then repeated and the small molecules ana-
lyzed by HPLC subsequent to dephosphorylation. When we
started with a 1:1 ratio of [5-3H]F2CTP to RTPR, 0.71 equiv of
cytosine and 0.19 equiv of unreacted F2C were recovered. In
addition, 0.15 equiv of cytosine co-eluted with RTPR, accounting
for all of the radiolabel.
Identification of Peptide or Peptides of RTPR Labeled
during Its Inactivation with F2CTP and Insight into the
Structure of the Sugar Moiety or Moieties Responsible for
Labeling. As summarized in Table 1, 0.47 3H labels from
[10-3H]F2CTP was found covalently attached to RTPR and is
likely associated with the loss of 47% of the RTPR activity.
Subsequent to inactivation of RTPR by [10-3H]F2CTP, the
protein was denatured in 8 M guanidine and the cysteines were
alkylated with iodoacetamide. The alkylated RTPR was ex-
changed into the trypsin digest buffer [0.1 M NH4HCO3 (pH
8.2)] by Sephadex G-50 chromatography, and the protein-
containing fractions were pooled and analyzed for protein and
radioactivity. While 80% of the protein was recovered, only 0.14
equiv of radioactivity co-eluted with the protein, indicating
substantial loss of the label during the denaturation and iodo-
acetamide treatment. Furthermore, when the 3H-labeled RTPR
was digested with trypsin and the peptides were separated by
reverse-phase HPLC, 10% of the radioactivity eluted in the void
volume of the column and the remaining radioactivity eluted in a
very broad peak between 45 and 65 min (data not shown). These
results suggested that inactivation results in multiple sites of
labeling or more likely that the label is redistributed to multiple
residues during the workup.
Our previous experience with identifying the site of attachment
of the label to RTPR inactivated by the mechanism-based
inhibitor, [20-3H]-20-chloro-20-deoxynucleotide (Scheme 2), sug-
gested that the loss of label might be minimized by reduction with
NaBH4. Thus, subsequent to inactivation, the reaction mixture
was treated with 50 mM NaBH4. The labeled RTPR that was
recovered (Table 1) was reduced from 0.45 to 0.33 equiv. Under
the conditions of alkylation, trypsin digestion, and HPLC
purification of the resulting peptides, 0.22 equiv of radiolabel
was recovered. The results of a typical chromatography are
shown in Figure 1 where four main regions of radioactivity are
identified. The average total recovery of radiolabel from column
chromatography from six experiments was 74 ( 5%, with 20 (
2% in region I, 26 ( 4% in region II, 20 ( 5% in region III, and
21 ( 4% in region IV.
Covalent Labeling of RTPR by [10-3H]- and [5-3H]-
F2CTP. Our previous studies suggested that 1 equiv of F2CTP
was sufficient for 90% inactivation of RTPR within 30 s in the
presence and absence of reductants (TR, TRR, and NADPH),
and 95% by 2 min (21). At that time, we suggested that
inactivation might in part result from covalent labeling of RTPR
and in part from covalent labeling of C419 by a cobalamin
species (21). The availability of [10-3H]- or [5-3H]F2CTP has
now allowed us to test these proposals by tracking the fate of the
ribose ring in the case of [10-3H]F2CTP and the cytosine base in
the case of [5-3H]F2CTP. RTPR was incubated with these
compounds for 2 min, and the small molecules were separated
from the protein by SEC in the presence or absence of denatur-
ant. The results are summarized in Table 1. With [10-3H]F2CTP,
0.47 ( 0.02 equiv co-elutes with RTPR in both cases. With
[5-3H]F2CTP, only 0.16 equiv co-elutes with RTPR in the
presence and absence of reductants, with a slight reduction in
recovered label under denaturing conditions. The substoichio-
metric labeling, the >90% inactivation, and the observation that
C419 can be modified with the corrin of AdoCbl suggest a
branching mechanism for inactivation of RTPR (21).
Release of Cytosine Accompanies Inactivation. It was
surprising that there was significantly less cytosine bound to
RTPR than ribose (Table 1), as our earlier studies failed to detect
Fractions from each region of the chromatogram were pooled
and rechromatographed at pH 6.4 in potassium phosphate
buffer. The results from region I are shown in Figure 1B. Regions

Article
Biochemistry, Vol. 49, No. 7, 2010 1411
Table 1: Covalent Labeling of RTPR with [10-3H]- and [5-3H]F2CTP
enzyme
F2CTP label
TR, TRR, and NADPH
Sephadex G-50a
label eluting with protein (equiv/RTPR)
wild type
wild type
wild type
wild type
10-3H
10-3H
10-3H
10-3H
10-3H
5-3H
5-3H
5-3H
5-3H
5-3H
þ
-
-
-
-
þ
þ
-
-
-
native
0.46
0.47
0.49
0.44
0.33
0.17
0.13
0.15b
0.12
0.03
denaturing
native
denaturing
native
wild type, NaBH4 at 2 min
wild type
native
wild type
denaturing
native
wild type
wild type
denaturing
native
wild type, NaBH4 at 2 min
aUnder denaturing conditions, 6 M guanidinium hydrochloride was added to the inactivation mixture before it was loaded onto the Sephadex column and
the column was eluted with 2 M guanidinium hydrochloride. bUnder these conditions, the small molecules were dephosphorylated and analyzed by HPLC and
0.71 equiv of cytosine and 0.18 equiv of F2C were found.
Scheme 2: Proposal for Structures That Can Be Accommo-
dated by the Labeling Observed in the MS Data
F
IGURE 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) [10-3H]F2CTP with NaBH4, (B)
[10-2H,3H]F2CTP with NaBH4, and (C) [10-3H]F2CTP with NaBD4.
The features at 1102.5 and 1347.7 Da are not from RTPR, as MS/MS
fragmentation analysis showed no matches with sequences from
RTPR. The inset shows a zoom of spectrum A around the 2004 Da
peak to highlight the presence of a peak at 2020 Da.
distribution shifted such that the 1 Da peak increases in intensity
[Figure 2B, Figure 3A (II), and Figure 3B (II)], allowing
confirmation of the source of the label. Alternatively, the
experiments quenched with NaB2H4 will show a shift of x Da
over the peptides quenched with NaBH4 [Figure 2C, Figure 3A
(III), and Figure 3B (III)], where x indicates the number of
hydrogens in the label originating from the borohydride quench.
The MALDI spectra of the peptides isolated from region I
under the three sets of conditions are shown in Figures 2A-C
and 3A,B. Two peptides, one with a mass of 2004 Da and one
with a mass of 2063 Da, show shifts in isotopic distribution from
the nondeuterated material [compare Figure 3A (I) with
Figure 3A (II and III) and Figure 3B (I) with Figure 3B (II
and III)]. No peptides from regions II and III (Figure 1) showing
the shift in isotopic distribution were detected in the MALDI
analysis, despite these regions representing a substantial percen-
tage of the radioactivity recovered from the trypsin digests. We
propose the lack of identifiable labeled peptides is due to a
combination of a number of potential cross-linked species with a
low incidence of each specific sequence (see the discussion of the
mechanism below) and that some of these cross-linked peptides
might be too large to be seen in the high-resolution MALDI
experiments.
II-IV were also rechromatographed (data not shown). All label
was lost from region IV (45).
Analysis of the Labeled Peptide Isolated from Region I
by MALDI-TOF Mass Spectrometry. MALDI analysis was
performed on the peptides isolated from region I (Figure 2A-C
and Figure 3A,B) and regions II and III (data not shown). To
facilitate identification of peptides containing a sugar fragment
derived from F2CTP, two additional experiments were con-
ducted. In one set of experiments, [10-2H,3H]F2CTP with
∼60% 2H at the 10 position and a specific activity of 2600 cpm/
nmol was used for the inactivation, and in the second set of
experiments, NaB2H4 replaced NaBH4. Inactivation reactions
involving these changes will result in the same, labeled pep-
tides, but with additional mass and a predictable mass distri-
bution. When using F2CTP partially labeled with 2H, any
peptides incorporating the sugar ring of F2CTP show an isotopic

1412 Biochemistry, Vol. 49, No. 7, 2010
Lohman and Stubbe
of a ketone or the 1,4-reduction of an unsaturated ketone
(Scheme 2). In either case, the observed mass of the adduct
would result in the addition of 1 Da. In addition, the ketone
subsequent to 1,4-reduction could then itself be reduced, resulting
in the incorporation of a second deuterium (þ2 Da). The analysis
of peptides isolated subsequent to this treatment is shown in
Figure 2C, Figure 3A (III), and Figure 3B (III).
In the expansion of the 2004 Da peak (Figure 3A), the mass of
the peptide shifts from 2004 to 2005 Da after NaB[2H]4 trapping
[Figure 3A (I and III)]. In the expansion of the 2063 Da peak
(Figure 3B), the mass is shifted by 2 Da to 2065 Da [Figure 3B (I)
and Figure 3B (III)]. These results establish that one hydrogen in
the species with a mass of 2004 Da and two hydrogens in the
species with a mass of 2063 Da come from NaBH4.
Analysis by MS/MS. To gain more information about the
structure of the modification(s), the sequence of the peptide, and
the residues alkylated, MS/MS analysis was performed. The
initial focus was on the peptide with a mass of 2020 Da
(Figure 2A, inset) where the mass itself is consistent with two
cysteines modified by acetamide. This assignment was confirmed
by post-source decay (PSD) fragmentation (Figure S1 of the
Supporting Information). In this method, additional energy is
applied to the peptide of interest, initiating predictable fragmen-
tation patterns. The major site of fragmentation is at peptide
bonds and gives rise to two series of ions: the y series in which the
charge is retained on the N-terminus and the b series with the
charge retained on the C-terminus (Scheme S2 of the Supporting
Information) (51, 52). Each mass peak within a given series is thus
associated with a truncated peptide, and the difference in mass
between each peak within a given series is the mass of one amino
acid in the sequence. The relative abundance of each ion series
detected is highly peptide dependent, and in the case of the 2020
Da peptide (Figure S1 of the Supporting Information), the y
series dominates the spectrum. A number of additional peaks can
be seen that are -18 and -17 Da from the main y series. These
are the y0 and y* series, representing loss of water and ammonia,
respectively, from the y series ions. The peaks from the major “y”
series are marked (vertical line), and the difference in mass
indicates the amino acid lost, allowing sequencing of the peptide.
Most remaining peaks visible in the spectrum are b series
peptides. This spectrum allows N-terminal sequencing by viewing
the y series. Fourteen y ions are observable, showing mass
differences consistent with the C-terminal RTPR peptide, with
both cysteines modified by acetamide.
The MS/MS spectrum of the 2063 Da peptide shows the same
series of ions as in the doubly acetamide-labeled C-terminal
peptide (Figure S1 of the Supporting Information), but shifted by
þ43 Da (Figure 4). The same series is visible in the 2065 Da
peptide from the NaB2H4-quenched reaction, but at þ45 Da
(data not shown). Once the first cysteine is removed, both the
normal series seen in the MS/MS spectrum for the acetamide-
modified peptide [peaks at 831, 702, 645, 588, and 517 Da
(Figure 4)] and a series which remains at þ43 Da (þ45 Da for
the NaB2H4-quenched reaction) can be seen. After the cleavage
of the second cysteine, only the normal mass peak remains
(a fragment at 357 Da). The label derived from the sugar
fragment of F2CTP is thus attached to either C731 or C736. This
method is not quantitative, and nothing can be said about the
relative extent of labeling at each site. As predicted by MALDI,
the sugar fragment has a mass of 101 Da (43 Da larger than that
of acetamide) and incorporates two deuteriums when the in-
activation is quenched with NaB2H4.
F
IGURE 3: (A) Expansion of the MALDI spectrum in Figure 2 in the
region around the 2004 Da peak: (I) [10-3H]F2CTP with NaBH4, (II)
[10-2H,3H]F2CTP with NaBH4, and (III) [10-3H]F2CTP with NaBD4.
(B) Expansion of the MALDI spectrum in Figure 2 in the region
around the 2063 Da peak: (I) [10-3H]F2CTP with NaBH4, (II)
[10-2H,3H]F2CTP with NaBH4, and (III) [10-3H]F2CTP with NaBD4.
Our previous studies with RTPR inactivated by [20-3H]-20-
chloro-20-deoxy-UTP followed by NaBH4 trapping identified
alkylation of cysteines within the C-terminus of RTPR (46). The
C-terminal tail contains a pair of cysteines (C731 and C736
)
essential to the function of the enzyme, re-reducing the active
site disulfide formed after every nucleotide reduction (47-50).
The expected mass of this C-terminal peptide with both cysteines
[C731 and C736 within the peptide of residues 722-739
(D722LELVDQTDCEGGACPIK739)] labeled with acetamide is
2019.90 Da and should be present in our digests if this region is
alkylated. This peptide is detected at 2020.04 Da (Figure 2, inset)
and serves as an internal control for the identification of
C-terminal peptides labeled with F2CTP derivatives.
Since one or both of these cysteines could be the site of
alkylation, the labeled peptide could be missing one or both
acetamides [loss of one or two 58 Da adducts gives rise to 2020 -
2 (58) = 1904 Da or 2020 - 58 = 1962 Da species]. If both
acetamides are missing, a sugar moiety from F2CTP would need
to have a mass of 100 Da (1904 þ 100 = 2004) to give the
observed mass of 2004 Da. Alternatively, if the peptide at 2063
Da corresponds to the C-terminal peptide with one acetamide,
then the group derived from F2CTP would have a mass of 101 Da
(1904 þ 58 þ 101 = 2063).
To learn more about the structure of the labeled species and
the number of hydrogen atoms derived from NaBH4, the
inactivation mixture with [10-3H]F2CTP and NaB2H4 (98% D)
was examined. Trapping with NaB2H4 can occur by 1,2-reduction

Article
Biochemistry, Vol. 49, No. 7, 2010 1413
F
IGURE 4: (A) MS/MS spectrum of the 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 molecular masses detected are shifted þ43 Da relative to the
acetamide-labeled C-terminal peptide (Figure S1 of the Supporting Information), suggesting alkylation at cysteine by a group 43 Da 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).
F
IGURE 5: MS/MS spectrum of the peak at 2004 Da corresponding to the C-terminal peptide of RTPR (DLELVDQTDCEGGACPIK)
containing an internal cysteine-cysteine cross-link derived from F2CTP. The y ion series is indicated. No regular ions were detected in the mass
range of 360-970 Da.
The MS/MS spectrum of the major labeled peak at 2004 Da is
more complex than that at 2063 Da (Figure 5). The expected y
series for the C-terminal peptide is still evident, -16 Da relative to
the diacetamide-modified C-terminal peptide (Figure S1 of the
Supporting Information). Each of these peaks shows several
closely related series, fragments that are -17 or -18 Da relative
to each y ion (the y* series that has lost ammonia and the y0 series
that has lost water) and often show a fragment at -35 Da,
representing the loss of both water and ammonia. It is unclear
why the intensities of these secondary fragments are enhanced
relative to those of the corresponding series in the 2020 and 2063
Da (Figure 4) peptides. Additionally, after the loss of the
aspartate before C731 (giving the peak at 975 Da), the peaks
visible until the loss of the second cysteine do not correlate with
any simple mass series. This result indicates some irregular
fragmentation of the peptides in this mass range. The nature of
this fragmentation is not readily apparent. The final tripeptide
fragment after cleavage of C736 (PIK) is the same in this spectrum
as in those for the 2020 and 2063 Da species. The PSD for the
2005 Da peak in the NaB2H4 spectra is similar, but shifted þ1 Da
relative to the 2004 Da spectra, and the primary y series is of even
lower abundance relative to the y0 and y* series (data not shown).
These data are consistent with modification at C731 and C736. If
both acetamides are missing, a fragment with a mass of 100 Da
derived from F2CTP would account for the observed mass
(2020 - 2 ꢀ 58 þ 100 = 2004). Our interpretation of this data
is that this modification is a 100 Da fragment that is covalently
modifying both cysteines, providing an internal cross-link. Clea-
vages in the backbone between the two cysteines would not
actually fragment the peptide if they were connected through a
side chain cross-link and could account for the lack of regular
fragments in this region.
Interpretation of MALDI and MS/MS Data. The pep-
tides associated with masses at 2004 and 2063 Da represent
cysteine-modified C-terminal peptides of RTPR. Modification is
evident at both C731 and C736. The mass at 2004 Da is consistent

1414 Biochemistry, Vol. 49, No. 7, 2010
Lohman and Stubbe
with the replacement of both acetamides with a fragment of 100
Da that is covalently linked to both cysteines. The peptide at 2063
Da is consistent with the same peptide; however, it has one
cysteine labeled with acetamide and one with a sugar fragment of
101 Da. The NaB2H4-quenched sample shows that one hydrogen
on the 100 Da fragment comes from NaBH4 and that two on the
101 Da fragment come from NaBH4.
Scheme 2 outlines a model consistent with these data. A
furanone equivalent could react with the C-terminal cysteines
though conjugate addition, either with one cysteine leaving a
compound with an R,β-unsaturated ketone or with both cysteines
giving rise to a saturated ketone. Reduction with NaBH4 would
provide the structure with the correct mass and number of
hydrogens delivered from NaBH4. Other alternative schemes
that can be envisioned are inconsistent with the NaB2H4 labeling
experiment.
Change in the Conformation of RTPR Subsequent to
Labeling by F2CTP Determined by SDS-PAGE. Recent
studies with of class Ia RNRs inactivated by F2CDP and
analyzed by SDS-PAGE subsequent to inactivation revealed
that ∼30% of the R migrated as a protein with a larger molecular
mass (23-25). Inactivation reaction mixtures of RTPR were
therefore analyzed by SDS-PAGE to detect any changes in
conformation related to the inactivation process (Figure 6).
Samples with and without DTT were analyzed at 0 and 5 min
subsequent to the inactivation with and without boiling of the 5
min sample. All inactivations quenched at 5 min showed the
presence of a new major band at ∼100 kDa representing 25% of
the overall intensity, with a corresponding reduction in the
intensity of the wild-type RTPR band at 75 kDa. Assuming
75-80% of the RTPR is active (47, 53-55), this new feature
represents ∼33% of the RTPR. We suggest that it is an altered
protein conformation that is generated regardless of the presence
or absence of DTT in the assay buffer or whether the sample is
boiled prior to sample loading. Samples at 5 min also showed a
second new minor band (<5% intensity).
F
F2CTP.
IGURE 6: SDS-PAGE of RTPR inactivated by treatment with
DISCUSSION
We previously reported SF kinetic experiments for the inacti-
vation of RTPR with AdoCbl by F2CTP. The results demon-
strated that 0.7 equiv/RTPR of cob(II)alamin was formed with a
kobs of 50 s-1. RFQ EPR experiments further revealed that
cob(II)alamin was exchange coupled to a thiyl radical (1.4 spins/
RTPR) and that over the next 255 ms the amount of total spin
remained unchanged and a new radical that was weakly exchange
coupled to cob(II)alamins was generated. We now propose that
this initially formed radical is 13 (Scheme 3) and that it partitions
into two major pathways (Scheme 3A,B). Pathway A results in
covalent modification of RTPR by the sugar moiety of F2CTP
and is the focus of this paper, while pathway B results in the
production of 0.25-0.3 equiv of a stable cytidine nucleotide
radical and is the focus of the preceding paper (27). These two
pathways account for 0.75-0.8 equiv of F2CTP consumed in the
inactivation study, which is sufficient for complete RTPR
inactivation, given that our previous pre-steady state experiments
have indicated that only 75-80% of our recombinant protein is
active (47, 53-55).
Similarites of the Mechanism of Inactivation of RTPR
by F2CTP and Class Ia (E. coli and human with β2 or
p53β2) RNRs by F2CDP. First, in all RNRs that we have
examined, inhibition by a gemcitabine nucleotide requires only a
single nucleotide per R for complete inactivation (24, 25). In class
I RNRs that have complex quaternary structures (Rnβm, where
n = 2, 4, or 6 and m = 2, 4, or 6), complete inactivation by one
F2CDP per R results from the increased affinity of the Rnβm
subunits because of the inactivation in one Rβ pair. Second,
despite the stoichiometry of the inhibition reaction, there are at
least two pathways responsible for inactivation: one involving
covalent modification and one involving destruction of the
cofactor (pathways A and B, respectively, in Scheme 3). Third,
there are ∼0.5 label from the sugar moiety of [10-3H]F2CTP that
becomes covalently attached to R. The attachment site(s) is
chemically labile, making identification of these sites challenging.
Fourth, in all cases, the sugar-modified R subunit of RNR
migrates on a SDS-PAGE gel as a protein with a higher
molecular mass than the unmodified RNR. Fifth, all RNRs
inactivated by F2CD(T)Ps are accompanied by release of
approximately one cytosine and the loss of two fluoride ions.
Inactivation of C119S-RTPR by [10-3H]F2CTP. Studies
of the class Ia RNRs suggested that an active site cysteine
involved directly in nucleotide reduction (C225 in E. coli RNR)
is alkylated by the sugar moiety of F2CDP (24, 25). C119S-
RTPR, a mutant of the corresponding R face cysteine implicated
in labeling in the class Ia RNR, was therefore inactivated by
[10-3H]F2CTP and analyzed by SEC. The results demonstrated
0.33 equiv of radiolabel co-eluting with the protein under
denaturing conditions. This number contrasts with the 0.47 equiv
labeling for the wild-type RTPR and suggests that this cysteine
might in part represent a site of labeling, not identified in the
peptide mapping analysis described above.
In a separate experiment, the small molecule fraction of the
inactivation mixture with C119S-RTPR was examined, subse-
quent to quenching at 2 min with NaBH4 to reduce any transient
ketone-containing species. The phosphates of the nucleotides
were removed with alkaline phosphatase, and the small molecules
were analyzed by RP-HPLC. Three regions of radioactivity were
identified: one broad region (15-19 min, 26% of the radio-
activity) and two sharper peaks (22.2 min, 43% of the radio-
activity, and 23.2 min, 11% of the radioactivity). The peak at 22.2
min co-eluted with 20-deoxy-20-fluorocytidine, while the peak at
23.3 min co-eluted with F2C. The presence of a compound with a
mass equal to that of 20-fluorocytidine was confirmed by high-
resolution ESI-MS for the 22.2 min peak. Trapping of this species
has interesting mechanistic implications discussed below.

Article
Biochemistry, Vol. 49, No. 7, 2010 1415
Scheme 3: Proposed Mechanism of Inactivation of RTPR by F2CTP by the Nonalkylative and Alkylative Pathways
Scheme 4: Proposed Mechanism for Generation of Furanone during the Inactivation of RTPR with F2CTP
The data, in sum, suggest that the mechanism of inactivation of
RNRs by Gemzar nucleotides does in fact follow the paradigm
for other 20-substituted nucleotide mechanism-based inhibitors
(Scheme 1). The loss of the second fluoride ion, however, makes
the mechanism of F2CD(T)P more complex than the generic
mechanism for the 20-monosubstituted deoxynucleotide mechan-
ism-based inhibitors (Scheme 1). However, we propose that the
predominant mechanism of loss of the second fluoride is also
likely to be similar in all RNRs (Scheme 3).
Proposed Mechanisms of Covalent Modification of
RTPR. The mechanism of covalent labeling of RTPR is com-
plex, and many possibilities have been considered. Our current
model in Scheme 3 proposes that 13 is common in all pathways.
Evidence in support of this intermediate comes from the trapping
of 20-deoxy-20-fluorocytidine subsequent to the inactivation of
C119S-RTPR with F2CTP using NaBH4. To account for the
majority of labeled RTPR (pathway A of Scheme 3), C119 or C408
is proposed to directly attack C20 of 13. This attack would
prevent escape of the sugar moiety from the active site and allow
the trapped sugar to reside in the active site long enough to lose
tripolyphosphate. Subsequent alkylation could then occur at C10
or C50 (16, Scheme 3), providing an explanation for our inability
to isolate well-behaved peptide adducts (Figure 1). In the class Ia
RNR inactivation studies, mutagenesis studies suggest that the
C119 equivalent is involved in covalent labeling (23-25). In the
case of RTPR, at most 30% of the covalent modification could
arise from this cysteine. Additional labeling could potentially
come from C408. In pathway B, instead of attack by C119 or C408
,
attack by an active site H2O is proposed, leading to 14. The
evidence that supports this proposal is described in the preceding
paper (27).
Our characterization of the radiolabeled C-terminal peptide of
RNR suggests that covalent modification is more complex than
that indicated in Scheme 3. A portion (10-15%) of the covalent

1416 Biochemistry, Vol. 49, No. 7, 2010
Lohman and Stubbe
labeling requires an additional pathway as the mass spectro-
metric data suggest that F2CTP is converted to a furanone
(Scheme 2) that alkylates one or both of the C-terminal cysteines
of RTPR. It is difficult to rationalize how these adducts could be
generated from 15 (Scheme 3). A proposal for the generation of
the furanone is shown in Scheme 4. This mechanism starts with
common intermediate 13. However, in this pathway, 13 is
reduced by a mechanism similar to that proposed for CTP
reduction, involving intermediates 17 and 18. This intermediate
can now lose HF to generate 19, which subsequent to electron
transfer from C408 would rapidly eliminate cytosine. Intermediate
20 can only be alkylated by the C-terminal cysteines as the active
site cysteines involved in nucleotide reduction are in a disulfide.
Once the first alkylation occurs, tripolyphosphate can eventually
be released and the second alkylation can occur.
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SUMMARY
The mechanism of F2CTP inactivation of RTPR is complex
even though it is stoichiometric. Perhaps most surprising is the
fact that the remarkable number of similarities in the mechanism
of inactivation shared between the class II RNR described in this
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ACKNOWLEDGMENT
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cleotide reductases. In Comprehensive Natural Products Chemistry
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We thank Dr. Aaron Hoskins for thoughtful review of the
manuscript and Dr. John Leszyk at UMass Medical School for
performance and assistance with interpretation of the mass
spectrometry experiments.
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SUPPORTING INFORMATION AVAILABLE
Outline of F2C synthesis (Scheme S1), depiction of PSD
peptide fragments (Scheme S2), and MS/MS analysis of the
2020 Da RTPR peptide (Figure S1). This material is available
free of charge via the Internet at http://pubs.acs.org.
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