Transition-state analysis of Trypanosoma cruzi uridine phosphorylase- catalyzed arsenolysis of uridine

Article abstract of DOI:10.1021/ja2031294

Uridine phosphorylase catalyzes the reversible phosphorolysis of uridine and 2′-deoxyuridine to generate uracil and (2-deoxy)ribose 1-phosphate, an important step in the pyrimidine salvage pathway. The coding sequence annotated as a putative nucleoside phosphorylase in the Trypanosoma cruzi genome was overexpressed in Escherichia coli, purified to homogeneity, and shown to be a homodimeric uridine phosphorylase, with similar specificity for uridine and 2′-deoxyuridine and undetectable activity toward thymidine and purine nucleosides. Competitive kinetic isotope effects (KIEs) were measured and corrected for a forward commitment factor using arsenate as the nucleophile. The intrinsic KIEs are: 1′-¹⁴C = 1.103, 1,3-¹⁵N ₂ = 1.034, 3-¹⁵N = 1.004, 1-¹⁵N = 1.030, 1′-³H = 1.132, 2′-²H = 1.086, and 5′-³H₂ = 1.041 for this reaction. Density functional theory was employed to quantitatively interpret the KIEs in terms of transition-state structure and geometry. Matching of experimental KIEs to proposed transition-state structures suggests an almost synchronous, S _N2-like transition-state model, in which the ribosyl moiety possesses significant bond order to both nucleophile and leaving groups. Natural bond orbital analysis allowed a comparison of the charge distribution pattern between the ground-state and the transition-state models.

Full text of DOI:10.1021/ja2031294

ARTICLE
pubs.acs.org/JACS
Transition-State Analysis of Trypanosoma cruzi Uridine
Phosphorylase-Catalyzed Arsenolysis of Uridine
Rafael G. Silva, Mathew J. Vetticatt, Emilio F. Merino,† Maria B. Cassera,† and Vern L. Schramm*
Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx,
New York 10461, United States
S Supporting Information
b
ABSTRACT: Uridine phosphorylase catalyzes the reversible
phosphorolysis of uridine and 20-deoxyuridine to generate uracil
and (2-deoxy)ribose 1-phosphate, an important step in the
pyrimidine salvage pathway. The coding sequence annotated as
a putative nucleoside phosphorylase in the Trypanosoma cruzi
genome was overexpressed in Escherichia coli, purified to
homogeneity, and shown to be a homodimeric uridine phos-
phorylase, with similar specificity for uridine and 20-deoxyur-
idine and undetectable activity toward thymidine and purine
nucleosides. Competitive kinetic isotope effects (KIEs) were
measured and corrected for a forward commitment factor using
arsenate as the nucleophile. The intrinsic KIEs are: 10-14C =
1.103, 1,3-15N2 = 1.034, 3-15N = 1.004, 1-15N = 1.030, 10-3H = 1.132, 20-2H = 1.086, and 50-3H2 = 1.041 for this reaction. Density
functional theory was employed to quantitatively interpret the KIEs in terms of transition-state structure and geometry. Matching of
experimental KIEs to proposed transition-state structures suggests an almost synchronous, SN2-like transition-state model, in which
the ribosyl moiety possesses significant bond order to both nucleophile and leaving groups. Natural bond orbital analysis allowed a
comparison of the charge distribution pattern between the ground-state and the transition-state models.
he kinetoplastid Trypanosoma cruzi, the etiologic agent of
Chagas disease, possesses all genes codifying the six
since KIEs originate primarily from differences in bond vibration
frequencies as a molecule goes from the ground state to the
transition state in the course of a chemical reaction.8,9 Transition-
state analyses utilizing KIEs and computational modeling have
been reported for both nonenzymatic and enzymatic ribosyl
transfer reactions involving purine10ꢀ12 and pyrimidine and
pyridine13ꢀ16 nucleosides and nucleotides. Some of these transi-
tion-state models served as blueprints for the design of transition-
state analogues that act as potent enzyme inhibitors.17,18
Here the T. cruzi protein annotated as a putative nucleoside
phosphorylase was expressed, purified to homogeneity, and
demonstrated to be a uridine phosphorylase by substrate speci-
ficity studies. The oligomeric state of the protein was assessed by
size-exclusion chromatography, and the substrate specificity was
determined by steady-state kinetics. A combination of competi-
tive KIEs measured with arsenate as nucleophile, isotope trapping
experiment, and density functional theory (DFT) was employed to
propose a transition-state model for the reaction catalyzed by
T. cruzi UP (TcUP). The bond order and charge distribution
differences between ground and transition states as well as the
implications of the model for the chemical mechanism of the
reaction are discussed.
T
enzymes involved in de novo pyrimidine biosynthesis,1 while
the salvage pathway had only been suggested based on observa-
tion of uridine phosphorylase (UP) activity in cell extracts.2
Recently, a putative nucleoside phosphorylase from the related
protozoan Trypanosoma brucei was demonstrated to be a homo-
dimeric UP, and a UP-specific region was identified and pro-
posed to provide a tool to identify UPs from amino acid sequence
alignments. The UP-specific region of the T. brucei genome was
identified in T. cruzi and annotated as a putative nucleoside
phosphorylase, which was then suggested to be a UP.3
Uridine phosphorylase (EC 2.4.2.3) catalyzes the reversible
phosphorolysis of (20-deoxy)uridine to form (2-deoxy)ribose
1-phosphate (R-1-P) and uracil4 (Scheme 1A), an important step
in the pyrimidine salvage pathway.5 The reaction equilibrium
favors nucleoside formation, and the equilibrium constant (Keq)
was calculated, using Haldane relationships, to be approximately
0.6.6 This enzyme has been classified as a member of the
nucleoside phosphorylase-I (NP-I) family, which includes en-
zymes that catalyze ribosyl transfer reactions, such as purine
nucleoside phosphorylase (PNP) and methylthioadenosine phos-
phorylase (MTAP), on the basis of a common R/β-subunit fold
and the inability to accept thymidine as substrate.7
Kinetic isotope effects (KIEs) have long been used to obtain
information about reaction transition-state structure and geometry,
Received: April 5, 2011
Published: May 20, 2011
r
2011 American Chemical Society
9923
dx.doi.org/10.1021/ja2031294 J. Am. Chem. Soc. 2011, 133, 9923–9931
|

Journal of the American Chemical Society
ARTICLE
Scheme 1. UP-Catalyzed (A) Phosphorolysis and (B)
Arsenolysis of Uridine
laser desorption/ionization time-of-flight mass spectrometry (MALDI-
TOF).
Oligomeric State Determination. The molecular mass of the
purified protein was estimated by gel-filtration chromatography using a
Sephacryl S-200 26/60 column (GE Healthcare). All samples were run
at 0.5 mL minꢀ1 in 100 mM of HEPES, pH of 7.5, at 4 °C. Bovine
thyroglobulin (670 000 Da), bovine γ-globulin (158 000 Da), chicken
ovalbumin (44 000 Da), horse myoglobin (17 000 Da), and vitamin B12
(1350 Da) (Bio-Rad) were employed as MW standards. The Kav value
for each protein was calculated with eq 1, where Ve is the elution volume,
Vt is the total column volume, and V0 is the void volume. Kav was plotted
against the logarithm of MW:
Kav ¼ ðVe ꢀ V0Þ=ðVt ꢀ V0Þ
ð1Þ
HPLC Method. The formation of pyrimidine or purine bases upon
nucleoside phosphorolysis was assessed by reverse-phase chromatogra-
phy, at room temperature, using an HPLC model 2487 (Waters) and an
analytical C18 Delta Pak column (Waters) pre-equilibrated with 10
column volumes of 50 mM of triethylamine:acetic acid, pH of 5.0. Each
sample was injected and run at 1.0 mL minꢀ1 in the same buffer, and
base and nucleoside elution were followed by the increase in absorbance
at the appropriate wavelength. A typical reaction mixture (300 μL)
contained 10 μM of TcUP, 100 mM of HEPES, pH of 7.5, 50 mM of
phosphate, and 200 μM of nucleoside. Reactions were allowed to
proceed for 45 min prior to HPLC analysis. Each nucleoside and the
respective base were run as standards to determine their retention time.
In control experiments, TcUP, nucleoside, and phosphate were omitted,
one at a time, from the reaction mixture.
Enzymatic Assay and Substrate Specificity of TcUP. All
assays were performed under initial rate conditions at 25 °C and
100 mM of HEPES, pH of 7.5, in 120 μL, unless otherwise stated.
The decrease in absorbance at 282 nm upon conversion of (20-deox-
y)uridine to uracil (Δε = 1600 Mꢀ1 cmꢀ1),22 in the presence of TcUP, in
either phosphorolytic or arsenolytic (Scheme 1A or B, respectively)
reactions, was monitored in an SX-20 stopped-flow spectrophotometer
outfitted with a mercuryꢀxenon lamp (Applied Photophysics). The
substrate specificity and apparent steady-state parameters of the enzyme
were determined by measuring TcUP activity in the presence of varying
concentrations of one substrate and fixed, saturating concentrations of
the other. In all cases, the concentration of TcUP was 20 nM. Each data
point is the average of at least 10 replicates measured on the stopped-
flow. The data were fitted to eq 2, where v is the initial rate, V is the
maximal velocity, A is the concentration of the varying substrate, and KM
is the Michaelis constant for the varying substrate:
’ MATERIALS AND METHODS
Materials. D-[1-3H]ribose, D-[1-14C]ribose, and D-[6-3H2]glucose
were purchased from American Radiolabeled Chemicals, Inc. D-[6-14C]glu-
cose was purchased from PerkinElmer, Inc. [1,3-15N2]Orotate and
deuterium oxide were obtained from Cambridge Isotope Laboratories,
Inc. Pyruvate kinase (PK), myokinase (MK), hexokinase (HK), glucose-
6-phosphate dehydrogenase (G6PDH), glutamic acid dehydrogenase
(GDH), 6-phosphogluconic acid dehydrogenase (6PGDH), and phos-
phoriboisomerase (PRI) were from Sigma-Aldrich. Alkaline phospha-
tase (AP) was from Roche. Ribokinase (RK) and phosphoribosyl-R-1-
pyrophosphate synthetase (PRPPase) were prepared as previously
described.19,20 UMP synthase (UMPS) and [3-15N]orotate were kind
gifts from, respectively, Keith Hazleton and Yong Zhang of this laboratory.
All other chemicals and reagents were obtained from commercial
sources and were used without further purification.
Expression and Purification of TcUP. The expression vector
pJexpress414 containing the DNA sequence annotated as a T. cruzi
putative nucleoside phosphorylase (GeneDB ID Tc00.10470535095-
69.100), with an N-terminal His tag-encoding sequence and optimized
codons for heterologous expression in Escherichia coli, was obtained
from DNA 2.0, Inc. The construct was transformed into E. coli BL21-
(DE3) RIPL cells (Novagen). The transformed cells were grown in
LuriaꢀBertani medium containing 100 μg mLꢀ1 ampicillin, at 37 °C, to
an OD600 nm of 0.5 and induced by addition of isopropyl-1-thio-β-D-
galactopyranoside to a final concentration of 1 mM. Cells were allowed
to grow for an additional 15 h period and harvested by centrifugation at
20 800 g for 30 min. All purification procedures were carried out at 4 °C.
Cells were resuspended and incubated for 30 min in 25 mL of buffer A
(20 mM TrisꢀHCl, 5 mM imidazole, 500 mM NaCl, pH of 7.9)
containing 0.2 mg mLꢀ1 of lysozyme, 0.05 mg mLꢀ1 of DNase I, and a
tablet of complete protease inhibitor cocktail (Roche), disrupted with a
French press and centrifuged at 48 000 g for 30 min to remove cell
debris. The supernatant was loaded onto a Ni-NTA column pre-
equilibrated with buffer A. The column was washed with 6 column
volumes of buffer A and 10 column volumes of buffer B (20 mM of
TrisꢀHCl, 50 mM of imidazole, 500 mM of NaCl, pH of 7.9). The
adsorbed material was eluted with 6 column volumes of buffer C (20 mM of
TrisꢀHCl, 150 Mm of imidazole, 500 mM of NaCl, pH of 7.9), analyzed
by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE),21 concentrated using a 10 000 Da molecular weight (MW)
cutoff Amicon ultrafiltration membrane, dialyzed against 4 L of 100 mM
HEPES pH of 7.5, and stored at ꢀ80 °C. Protein concentration was
determined spectrophotometrically at 280 nm using the theoretical
extinction coefficient (ε280) of 19 410 Mꢀ1 cmꢀ1 (http://expasy.org). The
protein was further characterized by trypsin digestion and matrix-assisted
v ¼ ðVAÞ=ðKM þ AÞ
ð2Þ
Synthesis and Purification of Isotopically Labeled Uri-
dines. [10-3H]UMP and [10-14C]UMP were enzymatically synthesized
at 37 °C from [1-3H]ribose and [1-14C]ribose, respectively (Scheme S1
in the Supporting Information). Each reaction mixture contained 1 mM
of ribose, 20 mM of phosphoenolpyruvate (PEP), 0.2 mM of ATP,
20 mM of MgCl2, 1.2 mM of orotate, 100 mM of KH2PO4 (pH 7.5),
50 mM of KCl, 1 mM of DTT, 0.2 U mLꢀ1 of PRPPase, 2 U mLꢀ1 of PK,
2 U mLꢀ1 of MK, 1 U mLꢀ1 of UMPS, and 1 U mLꢀ1 of RK.
For the synthesis of [50-3H2]- and [50-14C]UMP, D-[6-3H2]- and
D-[6-14C]glucose were, respectively, utilized as precursors (Scheme S1
in the Supporting Information). Each reaction mixture contained 1 mM
of glucose, 20 mM of PEP, 0.2 mM of ATP, 20 mM of MgCl2, 1.2 mM of
orotate, 140 mM of KH2PO4 (pH 7.5), 50 mM of KCl, 2.5 mM of DTT,
0.2 mM of NADPþ, 20 mM of R-ketoglutarate (R-KG), 5.5 mM of
NH4Cl, 0.2 U mLꢀ1 of PRPPase, 2 U mLꢀ1 of PK, 2 U mLꢀ1 of MK,
1 U mLꢀ1 of UMPS, 1 U mLꢀ1 of G6PDH, 1 U mLꢀ1 of 6PGDH,
9924
dx.doi.org/10.1021/ja2031294 |J. Am. Chem. Soc. 2011, 133, 9923–9931

Journal of the American Chemical Society
ARTICLE
0.5 U mLꢀ1 of PRI, 1 U mLꢀ1 of GDH, and 0.2 U mLꢀ1 of HK. Orotate
was replaced by [1,3-15N2]- and [3-15N]orotate for the synthesis of
[50-14C,1,3-15N2]- and [50-14C,3-15N]orotate, respectively. To synthe-
size [50-14C,20-2H]UMP, the reaction mix with D-[6-14C]glucose was
prepared in D2O. The deuterium was incorporated by the reaction
catalyzed by PRI, and the deuterium content in the final product was
determined by electrospray ionization mass spectrometry (ESI-MS).
Labeled UMPs, synthesized in 1 mL reaction volumes, were purified
by HPLC using the same method described above. The solvent was
evaporated by centrifugation under vacuum, and the pellet was dissolved
in water. The labeled UMPs were converted to labeled uridines via the
AP-catalyzed reaction, and the labeled uridines were purified with the
same protocol utilized for UMP purification.
Determination of Forward Commitment Factor. The for-
ward commitment (Cf) for uridine in the arsenolysis catalyzed by TcUP
was measured by the isotope trapping method.23 Pulse incubation
mixtures (20 μL) containing 20 μM of TcUP, 50 μM of [50-14C]uridine,
and 100 mM of HEPES, pH of 7.5, at 25 °C, were chased with a solution
(480 μL) consisting of 2 mM of uridine, 100 mM of HEPES, pH of 7.5,
and 1 mM of NaH2AsO4. After 5 s, the reactions were quenched with
50 μL of 1 N HCl and loaded onto charcoal columns (W. R. Grace &
Co.) pre-equilibrated with 10 mM ribose in 10% ethanol (v/v).
[5-14C]Ribose was eluted with 3 mL of 10 mM ribose in 10% ethanol
(v/v), dried by centrifugation under vacuum, and dissolved in 100 μL of
water and 10 mL of scintillation fluid (PerkinElmer). Radioactivity was
counted for 10 min in a Tricarb 2910 TR scintillation counter
(PerkinElmer). The values obtained in the absence of arsenate were
used as background to correct the others. The final value is the average of
eight replicates in two independent experiments. Cf was calculated with
eq 3, where Cf is the forward commitment, and Y is ratio of moles of
ribose to moles of TcUPꢀuridine complex. The concentration of
TcUPꢀuridine complex was calculated according to eq 4, where ES
is the concentration of the TcUPꢀuridine complex, E is the total
concentration of enzyme in the pulse, S is the total concentration of
uridine in the pulse, and KM is the Michaelis constant for uridine.
Experimental KIEs were calculated and extrapolated to 0% reaction
using eq 7, where Rf and R0 are the ratios of heavy to light isotopes at
partial and complete conversions, respectively, and f is the fraction of
substrate conversion.
KIE ¼ lnð1 ꢀ fÞ=ln½1 ꢀ f ꢁ ðRf =R0Þꢂ
ð7Þ
Computational Methods. The nucleophilic substitution reaction
involving arsenate and uridine was studied by density functional theory
(DFT) in B3LYP and using a 6-31G** basis set as implemented in
Gaussian 09.24 All ground-state structures were located as global
minima, and frequency calculations performed on the optimized struc-
tures contained no imaginary frequencies. All located transition struc-
tures, with and without geometric constraints, possessed only one
calculated imaginary frequency, a characteristic of first-order saddle
points that correspond to true transition states.25 The isotope effects for
each of the theoretical transition structures were calculated from scaled
theoretical vibrational frequencies using ISOEFF98.26 Tunneling cor-
rections were applied using a one-dimensional infinite parabolic barrier
model.27
’ RESULTS AND DISCUSSION
Purification, Activity, And Oligomeric State of TcUP. The
N-terminal His-tagged enzyme was successfully overexpressed
and purified to homogeneity. MALDI-TOF-TOF mass spectro-
metry analysis confirmed that the amino acid sequence of the
homogeneous protein was that annotated as a putative nucleo-
side phosphorylase from the T. cruzi genome.
In reaction mixtures containing either uridine or 20-deoxyur-
idine and phosphate, uracil was formed (Figure 1). No nucleo-
base formation was detected when reaction mixtures contained
thymidine, cytosine, orotidine, or any of the purine nucleosides.
Controls without phosphate eliminated a hydrolytic reaction for
uracil formation under conditions that would have detected a rate
10ꢀ6 that of phosphorolysis. Thus TcUP is a (20-deoxy)uridine-
specific uridine phosphorylase that does not catalyze nucleoside
hydrolysis in the absence of phosphate, as do some purine and
pyrimidine phosphorylases.28,29 This is in agreement with the
specificity proposed for most UP members of the NP-I family7
but contrasts with the detected, though small, activity toward
thymidine reported for T. brucei UP.3 The lack of hydrolysis of
uridine by TcUP may also relate to the chemical stability of its
N-ribosidic bond, which is stable to 1 N of HCl at 100 °C for
more than 3 days.30
Cf ¼ Y=ð1 ꢀ YÞ
ð3Þ
ES ¼ ðE ꢁ SÞ=ðS þ KMÞ
ð4Þ
Measurement of KIEs. All KIEs were measured by the competitive
radiolabel method,20 at 25 °C. A typical reaction mixture (1 mL)
contained 1 mM of NaH2AsO4, 10 nM of TcUP, 100 mM of HEPES,
pH of 7.5, and 100 μM of uridine (3H- and 14C-labeled and cold carrier).
The arsenolysis was allowed to proceed to 15ꢀ20% completion, then
half of the reaction mixture was loaded onto charcoal columns (Grance).
The remainder of the reaction was completely converted to products by
addition of 4 μM of TcUP and loaded onto charcoal columns. Column
pre-equilibration and elution steps as well as sample preparation for
scintillation counting followed the same procedures described for the
isotope trapping experiment. A sample with only [14C]uridine was also
counted as a standard. At least 10 replicates, in 2 independent experi-
ments, were averaged for each KIE.
Molecular sieve chromatography estimated the molecular
weight of TcUP to be 76 000 ( 2280 Da. The calculated
MW of the subunit, based on the amino acid sequence of the
His-tagged protein, is 38 630 Da (http://expasy.org), suggesting
that TcUP is a homodimer in solution. The same oligomeric state
has been proposed for T. brucei UP.3
Substrate Specificity and Apparent Kinetic Parameters.
The kinetic parameters for TcUP and uridine are in the range
reported for E. coli UP6 and human UP (Table 1).31 The
specificity of TcUP for uridine and 20-deoxyuridine is compar-
able, evidenced by the similar values of the specificity constants
(kcat/KM). Thus, the ribosyl 20-OH group is not essential for
substrate binding or catalysis. TcUP also accepts arsenate as nucle-
ophile, as is observed for other nucleoside phosphorylases.11,12,15
Commitments to Catalysis. Cf, the forward commitment, is
the probability of a substrate to proceed forward to catalysis, after
the Michaelis complex is formed, as opposed to dissociating to
free substrate. The reverse commitment (Cr) reflects the tendency
3
Samples were counted in dual-channel fashion, with the H signal
appearing only in channel 1 and the 14C signal in both channels. The
initial ratio of 3H to 14C counts per minute (cpm) was 2:1, and enough
material was used so that at least 10 000 cpm of 14C was obtained in
channel 2. The total 3H signal was assessed by eq 5, and the total 14C signal
by eq 6, in which 3H is the total number of cpm for this isotope, 14C is the
total number of cpm for this isotope, channels 1 and 2 are the number of
cpm in each channel, and r is the channel 1 to 2 ratio of 14C standard.20
3H ¼ channel 1 ꢀ ðchannel 2 ꢁ rÞ
14C ¼ channel 2 ꢁ ð1 þ rÞ
ð5Þ
ð6Þ
9925
dx.doi.org/10.1021/ja2031294 |J. Am. Chem. Soc. 2011, 133, 9923–9931

Journal of the American Chemical Society
ARTICLE
expression, and knowing their values permits calculation of
intrinsic KIEs.34 A Cf value of 0.283 ( 0.012 was measured for
uridine with TcUP, using arsenate as a nucleophile. This value is
similar to those reported for His257Phe (0.300 ( 0.010)35 and
Lys22Glu-His104Arg (0.239 ( 0.025)36 mutants of human PNP
and for Val39Thr-Asn123Lys-Arg210Gln mutant of bovine PNP
(0.243 ( 0.026)37 and permits correction of experimental KIEs
to yield intrinsic values. The isotope trapping experiment estab-
lishes that uridine can bind to free enzyme, leading to a catalytically
competent complex upon arsenate binding. The result eliminates
a mandatory ordered kinetic mechanism in which the nucleo-
phile binds to the free enzyme.
Experimental and Intrinsic KIEs. Competitive KIEs mea-
sured for the TcUP-catalyzed arsenolysis of uridine and their
corresponding position and type are shown in Table 2. In
enzymatic reactions, the competitive method provides isotope
effects on V/K, therefore reporting on steps starting with free
substrate up to and including the first irreversible step.38 Quan-
titative information on the transition-state structure can only be
gathered from intrinsic KIEs, the ones reporting on the micro-
scopic chemical step of the enzymatic reaction.33 Experimental
KIEs can be corrected for Cf and Cr to yield intrinsic ones,
according to eq 8, where LV/K is the experimental KIE on V/K,
L
Lk is the intrinsic KIE, and Keq is the isotope effect on the
equilibrium constant.39
L
L
LV =K ¼ ð k þ Cf þ Keq ꢁ CrÞ=ð1 þ Cf þ CrÞ ð8Þ
Arsenate is used to replace phosphate in studies of nucleoside
phosphorylase reactions inasmuch as the product ribose 1-
arsenate is rapidly hydrolyzed to ribose. This renders the overall
reaction irreversible and reduces eq 8 to 9, as Cr is negligible.11
Accordingly, intrinsic KIEs for the TcUP-catalyzed reaction were
obtained upon correction for Cf (Table 2). Further evidence that
Cf is the only term reducing the experimental KIEs from their
intrinsic values is provided by the 1-15N KIE, which, after correction
for Cf, is equal, within experimental error, to the maximum value
found computationally for any possible mechanism or transition-
state structure of this reaction (discussed below).40
Figure 1. HPLC elution profile of TcUP-catalyzed reactions. (A)
Uridine, 20-deoxyuridine, and uracil run as standards. (B) Phosphor-
olysis of uridine. (C) Phosphorolysis of 20-deoxyuridine.
L
LV =K ¼ ð k þ Cf Þ=ð1 þ Cf Þ
ð9Þ
The large value of the 10-14C KIE and the relatively modest
value of the 10-3H KIE suggest a bimolecular ANDN mechanism41
with an SN2-like transition state. In nucleophilic substitution
reactions of nucleosides, large primary 14C KIEs occur only with
significant bond order between the developing oxocarbenium ion
and both the leaving group and the attacking nucleophile at the
transition state. The relatively small R-secondary 3H KIE indicates
incomplete sp3 to sp2 rehybridization at the transition state.42
The large 1-15N KIE is consistent with extensive, but not full, 10-
C to1-N bond loss at the transition state, with some bond order
remaining,43 as indicated by the 10-14C KIE. The large 20-2H KIE
suggests significant hyperconjugation between the 20-Cꢀ20-H σ-
bond and the developing vacant 10-C p-orbital, forming a partial
10-Cꢀ20-C π-bond.44 As the 20-Cꢀ20-H bond is weakened by
electron density loss, the β-secondary 2H KIE increases.13 The
isotope effects eliminate a steady-state ordered kinetic mecha-
nism in which uridine is the first substrate to bind to the enzyme,
as saturating arsenate concentration in this mechanism would
decrease the KIEs to unity.45 Interpretation of the intrinsic KIEs
Table 1. TcUP Apparent Steady-State Kinetic Parameters
varying substrate
kcat (sꢀ1
)
KM (μM)
kcat/KM (Mꢀ1 sꢀ1
)
uridine
12 ( 1
21 ( 2
298 ( 23
32 ( 4
5.7 ꢁ 105
4.0 ꢁ 104
4.7 ꢁ 105
3.9 ꢁ 104
3.0 ꢁ 105
3.3 ꢁ 104
phosphate
20-deoxyuridine
phosphate
uridine
15 ( 1
6 ( 1
385 ( 41
20 ( 2
arsenate
183 ( 17
for the first enzymeꢀproduct complex following the isotope-
sensitive step to undergo catalysis in the reverse direction instead
of moving forward to free product and enzyme through the first
irreversible step.32 Cf always refers to the substrate carrying the
isotopic label when dealing with competitive KIEs,33 uridine in
the present study. Significant commitment factors obscure KIE
9926
dx.doi.org/10.1021/ja2031294 |J. Am. Chem. Soc. 2011, 133, 9923–9931

Journal of the American Chemical Society
ARTICLE
Table 2. Kinetic Isotope Effects for the TcUP-Catalyzed Arsenolysis of Uridine
heavy uridine
light uridine
type of KIE
experimental KIE
intrinsic KIE
predicted KIE
10-3H
10-14C
50-14C,20-2H
50-14C,1,3-15N
50-14C,3-15
50-14C,1-15
50-3H2
50-14Ca
50-3H2
50-3H2
50-3H2
50-3H2
ꢀ
R-secondary
1.103 ( 0.004
1.080 ( 0.003b
1.067 ( 0.003b,c
1.027 ( 0.003b
1.003 ( 0.002b
1.024 ( 0.004d
1.032 ( 0.002
1.132 ( 0.005
1.103 ( 0.004
1.086 ( 0.004
1.034 ( 0.004
1.004 ( 0.003
1.030 ( 0.005
1.041 ( 0.003
1.126
1.109
1.080
1.027
1.002
1.025
1.058
primary
β-secondary
primary and β-secondary
β-secondary
primary
N
N
50-14C
δ-secondary
a The remote 50-14C KIE is assumed to be unity. b Experimental values corrected for remote 50-3H2 KIE according to the equation KIE = KIEobs
ꢁ
50-3H2 KIE. c Experimental values corrected for 2H content according to the equation KIE = (KIEobs ꢀ 1 þ F)/F, where F is the fraction of 2H in the
substrate. d Experimental values corrected for 3-15N KIE according to the equation KIE = KIEobs/3-15N KIE.
Figure 2. Stick models of DFT (B3LYP/6-31þG**)-optimized structures of (A) ground and (B) transition states. Carbon atoms are shown in gray,
oxygen in red, hydrogen in cyan, nitrogen in blue, and arsenic in green. The dashed lines (black) represent rCꢀN and rCꢀO, and the dotted lines (blue)
represent the distance between hydrogen and H-bond acceptor atoms. The full H-bond distances are described in the text.
in terms of transition-state structure was attempted using DFT-
based modeling.
Scheme 2. General Transition State for TcUP-Catalyzed
Arsenolysis of Uridine
Ground-State and Transition-State Search. The lowest-
energy reactant geometry was identified by optimizing uridine
structures with distinct conformations of the ribosyl ring and
orientations of uracil (coordinates for reactant structures are
available in the Supporting Information). A structure with a 30-
exo ribosyl pucker and the 2-O of uracil poised to accept a
hydrogen-bond (H-bond) from the 50-OH was found to be the
global minimum (Figure 2A). The choice of appropriate starting
material is essential for accurate prediction of KIEs, as they reflect
changes in bonding environment of atoms in the transition state
relative to the lowest-energy initial material free in solution.13
A systematic search for transition structures was conducted by
varying the distances between 10-C and 1-N (rCꢀN) and 10-C and
the nucleophilic oxygen of arsenate (rCꢀO) (Scheme 2) by 0.2 Å
increments. KIEs were calculated for every optimized structure
and compared with intrinsic values (coordinates and predicted
KIEs for all transition structure geometries are available in the
Supporting Information). The geometry that best reproduced
the intrinsic KIEs, particularly the distinct, large 10-14C KIE, was
further analyzed, as the primary carbon KIE is the most informa-
tive one for investigation of transition-state structure and geo-
metry of ribosyl-transfer reactions.46
systematically modified in terms of rCꢀN and rCꢀO, with incre-
ments of 0.1 Å and of 50-Oꢀ50-Cꢀ40-Cꢀ40-O and of 20-Cꢀ10-
Cꢀ1-Nꢀ2-C dihedral angles, and reoptimized. After calculation
of KIEs for each structure, the best matches were selected upon
comparison with intrinsic values (coordinates and predicted KIEs
for transition structure geometries with varying dihedrals are
available in the Supporting Information). An orientation with a
Enzymatic Transition Structures and Predicted KIEs.
The transition structures selected from the initial search were
9927
dx.doi.org/10.1021/ja2031294 |J. Am. Chem. Soc. 2011, 133, 9923–9931

Journal of the American Chemical Society
ARTICLE
H-bond between 50-OH and 1-N yielded calculated KIEs in
excellent agreement with intrinsic values and was stable enough
to be maintained with different rCꢀN and rCꢀO even after the
constraint on the 50-Oꢀ50-Cꢀ40-Cꢀ40-O dihedral angle was
removed.
Table 3. Bond Distances and Pauling Bond Orders in the
Ground-State and Transition-State Structures of TcUP-
Catalyzed Reaction
bond distance bond distance bond order bond order
in ground
state (Å)
in transition
state (Å)
in ground in transition
The orientation of uracil was important for accurate prediction
of the 20-2H KIE. However, all transition structures that provided
calculated KIE values in accordance with intrinsic ones were
characterized by two imaginary frequencies, indicating that the
structures were not located in first-order saddle points.25 To
overcome this difficulty, the side chain of an amino acid residue
was added to the model. This approach has recently been success-
fully employed to model the transition states of the reactions
catalyzed by human thymidine phosphorylase (TP)15,29 and
Phe200Gly mutant of human PNP.47 As a three-dimensional
structure of TcUP is not yet available, the crystal structure of
T. brucei UP3 was utilized to identify a residue that contacts uracil.
The two enzymes share 100% amino acid sequence identity in
the active site, and the chosen residue, glutamine (Gln) 248
(Gln249 in TcUP) is conserved in all uridine phosphorylases
characterized.3 The glutamine side chain was inserted in the
transition-state model to stabilize the anionic uracil leaving group
via H-bond contacts with the 2-O and 3-N of uracil anion,
similarly to the crystal structure.3 This orientation of uracil, with
the 2-O directed away from the 50-OH and 20-H, provided a good
match of the 20-2H KIE. The final transition structure
(Figure 2B) possesses a 20-Cꢀ10-Cꢀ1-Nꢀ2-C dihedral angle
of 85° and is characterized by a single imaginary frequency
corresponding to motion along the reaction coordinate, and its
calculated KIEs provide the best match for the intrinsic values
(Table 2).
In the model represented in Figure 2B, rCꢀN = 2.1 Å and
rCꢀO = 2.3 Å. Thus, TcUP-catalyzed arsenolysis of uridine
proceeds via an almost synchronous ANDN transition state with
moderate oxocarbenium ion character. This is in contrast with
the dissociative transition-state models, with no significant
participation of the attacking nucleophile, reported for inosine
arsenolysis by bovine, malarial, and human PNP11,46 and for the
hydrolytic cleavage of the N-glycosidic bond of 20-deoxyuridine
in DNA, catalyzed by E. coli uracil DNA glycosylase (UDG),48
while in agreement with the almost synchronous, though less
tight, transition-state structure proposed for the NADþ hydro-
lysis catalyzed by diphtheria toxin.43 SN2 mechanisms have also
been reported for E. coli UP phosphorolysis49 and for human TP-
catalyzed arsenolysis of thymidine, however in the latter, bond
breaking was significantly more advanced than bond making at
the transition state.15
bond
statea
statea
10-Cꢀ1-N
10-CꢀO(As)
10-Cꢀ40-O
10-Cꢀ20-C
10-Cꢀ10-H
20-Cꢀ20-H
40-Cꢀ40-O
50-Cꢀ50-O
40-Cꢀ50-C
1-Nꢀ2-C
1.47
2.10
2.30
1.31
1.53
1.04
1.10
1.48
1.42
1.52
1.37
1.36
1.39
1.41
1.00
ꢀ
0.35
0.25
1.49
1.03
1.18
0.98
0.92
1.03
1.07
1.40
1.44
1.31
1.22
1.41
1.55
1.09
1.09
1.45
1.41
1.53
1.40
1.38
1.38
1.41
1.07
0.98
1.00
1.00
0.97
1.07
1.03
1.27
1.35
1.35
1.22
1-Nꢀ6-C
3-Nꢀ2-C
3-Nꢀ4-C
a Bond orders >1 were calculated according to eq 10, while those
<1 were calculated according to eq 11.
distortion of the ribosyl pucker are features observed in other
enzymatic N-ribosidic cleavage reactions and are proposed to be
a strategy to help stabilize the charge development in the ribosyl
moiety at the transition state.11,43,48,50
Pauling Bond Orders and Charge Distribution at the
Transition State. Pauling bond order (niꢀj) is an exponential
function of interatomic distance and represents the number of
electron pairs shared by atoms i and j in a chemical bond.51
Comparison of niꢀj values for bonds in the reactant state and the
transition-state structures provides quantitative information on
the geometry and hybridization of a molecule along the reaction
coordinate. Accordingly, Pauling bond orders for specific bonds
in uridine and in the transition structure were calculated using
eq 10, for multiple bonds (niꢀj > 1), or eq 11, for partial bonds
(niꢀj < 1), where nij is the bond order between atoms i and j, rb is
the bond length for a single bond between atoms of elements
i and j, and rij is the bond length between atoms i and j.52
ꢀ rijÞ=0:3
nij ¼ eðr
ð10Þ
b
ꢀ rijÞ=0:6
nij ¼ eðr
ð11Þ
b
0
0
Bond orders (Table 3) are significant (nC1 ꢀN1 and nC1 ꢀO(As)
of 0.35 and 0.25, respectively) at the transition state, in agree-
ment with the large intrinsic 10-14C and 1-15N KIEs. Since niꢀj is a
nonlinear function of bond distance, a small difference in length
between bonds being broken and formed at the transition state
(0.2 Å) reflects a significant difference in bond order. The gain in
A comparison of substrate and transition structures highlights
the differences in forming the transition state. In reactant uridine,
the 50-OH group is oriented in a syn configuration, and the
distance between the ribosyl 50-O and the uracil 2-O is 2.82 Å,
allowing an intramolecular H-bond (Figure 2A). At the transition
state, the 50-OH group is in a syn configuration but is positioned
to form a H-bond with the leaving group 1-N, with a 50-Oꢀ1-N
distance of 3.18 Å, while the carbonyl 2-O is turned away from the
ribosyl plane and interacts with the amino group of the glutamine
side chain (Figure 2B). The ribosyl group of uridine has a 30-exo
and 20-endo pucker (Figure 2A) and assumes a flatter conforma-
tion at the transition state, with a less pronounced 30-exo pucker
(Figure 2B). This conformation favors hyperconjugation be-
tween the 20-Cꢀ20-H bond and the partially unoccupied 10-C
p-orbital.44 The syn configuration of the 50-OH group and the
0
0
0
0
nC1 ꢀC2 and the loss in nC2 ꢀH2 as the reaction reaches the
transition state agrees with hyperconjugation in the ribosyl
moiety,13 as discussed above, and explains the significant intrinsic
20-2H KIE. Incomplete sp2 hybridization of 10-C is indicated by
0
0
3
the nC1 ꢀO4 of 1.49, in conformity with the relatively modest
intrinsic 10- H KIE of 1.132. In comparison, nC1 ꢀO4 = 1.65 at
0
0
the transition state of human TP-catalyzed arsenolysis of thymi-
3
15
dine, with 10- H KIE = 1.177, and nC1 ꢀO4 = 1.71 at the
transition state of inosine arsenolysis catalyzed by the Phe200Gly
mutant of human PNP, for which 10-3H KIE = 1.254.47 These
0
0
9928
dx.doi.org/10.1021/ja2031294 |J. Am. Chem. Soc. 2011, 133, 9923–9931

Journal of the American Chemical Society
ARTICLE
Figure 3. NBO charge distribution in (A) ground and (B) transition states of TcUP-catalyzed reaction and in (C) the theoretical intermediates in case
the reaction proceeded by a stepwise mechanism. Negative charges are shown in red and positive charges in blue.
Scheme 3. Three General Nucleophilic Substitution Mechanisms Considered for TcUP-Catalyzed Arsenolysis of Uridinea
a In blue, DN*AN‡ stepwise mechanism; in green, ANDN concerted mechanism; in red, DN‡*AN stepwise mechanism. The double dagger denotes the
rate-limiting step.
two reactions proceed by highly asynchronous ANDN mech-
anisms, with more complete sp2 character in the ribosyl moiety of
their transition-state structures15,47 than in the nearly synchro-
nous ANDN mechanism reported here. A modest decrease in
9929
dx.doi.org/10.1021/ja2031294 |J. Am. Chem. Soc. 2011, 133, 9923–9931

Journal of the American Chemical Society
ARTICLE
nN3ꢀC2 is observed in the transition structure, consistent with the
normal intrinsic 3-15N KIE (Table 2). It is noteworthy that the
bond order analysis of the DFT-derived transition-state model
(Figure 2B) is in agreement with the qualitative interpretation of
the intrinsic KIEs discussed above.
A stepwise DN‡*AN mechanism,41,42 involving the rate-deter-
mining break of the N-ribosidic bond and a subsequent nucleo-
philic attack on the ribooxocarbenium intermediate (Scheme 3,
path in red), as described for bovine PNP-catalyzed arsenolysis of
inosine,11 was also considered. The largest calculated 10-14C KIE
(1.070) for this mechanism was significantly smaller than the
intrinsic value, ruling out this mechanism as a possibility. The last
mechanism modeled was a DN*AN mechanism featuring an
intermediate energetically similar to the transition state, with
comparable barrier heights for bond breaking and formation, as
proposed for the arsenolytic cleavage of inosine catalyzed by
human PNP, in which all measured isotope effects were EIEs
reporting on the formation of the ribooxocarbenium ion.46 This
mechanism is readily eliminated in the present work, since the
predicted 10-14C EIE was essentially unity, the 10-3H EIE was
three times as large as the intrinsic value, and the 50-3H2 EIEs
were inverse.
It should be pointed out that the term nucleophile-assisted
SN1 has been invoked to describe transition structures char-
acterized by very low bond orders to both the leaving group
and the latter, where the presence of the latter at the transition
state is mandatory to yield calculated KIEs that match experi-
mental values.57 Nonetheless, the bond orders in the transition
structure proposed for the TcUP reaction are significant, and
reactions with transition structures of comparable bond order
values to the ones presented here have widely been inter-
preted as SN2.15,42,55
Atomic charge distribution of reactants and transition states is
useful to understand catalytic strategies and transition-state analogue
design.53 Hence, natural bond orbital (NBO) charges were
calculated for the TcUP transition-state model and compared
with reactant uridine (Figure 3). As expected, the most conspic-
uous changes in NBO charges occur in the atoms along the
reaction coordinate with 10-C becoming more cationic and 1-N
more anionic, as the N-ribosidic bonding electrons move toward
uracil at the transition state. Concurrently, the attacking arsenate
oxygen is partially bonded and therefore less negative at the
transition state than in the reactant.47 A small reduction in
electron density occurs on 40-O, since it forms a partial π-bond
with 10-C, as evidenced by the nC1 ꢀO4 of 1.49. In the uracil
moiety, 5-C and 4-O accumulate extra electron density in the
transition structure, due to electron delocalization on the pyr-
imidine ring to stabilize the incoming N-ribosidic bond electrons.
The negative charges on the uracil moiety at the transition
structure may be slightly under-predicted in comparison with
uridine, owing to the stabilization from a H-bond with the
glutamine side chain. The glutamine amide donates a H-bond
(2.74 Å) to the leaving group 2-O, while its carbonyl oxygen
accepts a H-bond (3.15 Å) from the uracil 3-N (Figure 2B).
Similar H-bond patterns are observed between Gln248 and
product uracil in the T. brucei UP crystal structure.3
0
0
’ SUMMARY
NBO charges were also calculated for the theoretical inter-
mediates that would be formed if the reaction proceeded via a
stepwise, SN1 mechanism (Figure 3C). The most pronounced
differences occur in the fully formed ribooxocarbenium ion, in
which both 1-C and 4-O are significantly more deficient in
electron density as compared with their counterparts in the SN2-
like transition state for TcUP-catalyzed reaction (Figure 3B). This
attests to the relatively modest oxocarbenium ion character in the
transition state for this reaction, as suggested above by the
qualitative interpretation of the intrinsic KIEs.
The T. cruzi protein annotated as a putative nucleoside
phosphorylase is a (20-deoxy)uridine-specific homodimeric
uridine phosphorylase that accepts arsenate as an alternative to
phosphate as nucleophile. Intrinsic KIEs matched calculated
KIEs for a DFT-derived transition-state model in which both
the leaving group and the incoming nucleophile possess signifi-
cant bond order to the electrophile being transferred, with rCꢀN
=
2.1 Å and rCꢀO = 2.3 Å. The reaction proceeds via an ANDN
chemical mechanism in which uracil departs as a negative species
stabilized by interactions with the enzyme. The intrinsic KIEs are
fully compatible with an ANDN mechanism and are inconsistent
with other mechanisms for nucleophilic substitutions. Significant
changes in Pauling bond orders and NBO charge distribution
take place as the reaction proceeds from the ground to the
transition state. To the best of our knowledge, this is the first
report on the transition-state analysis of the reaction catalyzed by
uridine phosphorylase from any organism.
Other Chemical Mechanisms. The intrinsic KIEs obtained
here are consistent with a concerted, ANDN mechanism (Scheme 3,
path in green). However, elimination of alternative mechanisms
is also necessary. All bimolecular transition structures with a
protonated leaving group failed to yield theoretical KIEs that
matched the experimental values. Negatively charged nucleo-
bases have also been described in the transition-state models of
human and malarial orotate phosphoribosyl transferases,14,54
human TP,29 and uracil DNA glycosylase.48
‡
In stepwise DN*AN mechanisms,41,42 where the nucleobase
’ ASSOCIATED CONTENT
departs first, followed by the rate-limiting attack of the nucleo-
phile on the ribooxocarbenium ion (Scheme 3, path in blue),
the KIEs are the product of the equilibrium isotope effect
(EIE) for the first step and the KIE for the second step.29,55,56
For uridine arsenolysis, the predicted 1,3-15N2 EIEs were in
agreement with the intrinsic values for a negatively charged
uracil (1.027) but did not match the calculated values for the
departure of a neutral uracil with a proton on either 1-N
(1.008) or 4-O (1.020) positions. In all cases, the products of
the remaining EIEs and KIEs, for several rCꢀO, were incon-
sistent with the experimental results, including that for the
S
Supporting Information. Synthetic scheme for labeled
b
UMPs (Scheme S1) and geometries and calculated KIEs for
transition structures. This material is available free of charge via
the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
vern@aecom.yu.edu
Present Addresses
†Department of Biochemistry, Virginia Polytechnic Institute and
State University, Blacksburg, VA 24061, United States
10-14C KIE, where the largest predicted KIE for a DN*AN
‡
mechanism was 1.066.
9930
dx.doi.org/10.1021/ja2031294 |J. Am. Chem. Soc. 2011, 133, 9923–9931

Journal of the American Chemical Society
ARTICLE
’ ACKNOWLEDGMENT
Fox, D. J. Gaussian 09, revision A.1; Gaussian, Inc.: Wallingford, CT,
2009.
(25) Hirschi, J. S.; Takeya, T.; Hang, C.; Singleton, D. A. J. Am.
Chem. Soc. 2009, 131, 2397–2403.
(26) Anisimov, V.; Paneth, P. J. Math. Chem. 1999, 26, 75–86.
(27) Bell, R. P. The tunnel effect in chemistry; Chapman and Hall:
London, 1980.
(28) Kline, P. C.; Schramm, V. L. Biochemistry 1992, 31, 5964–5973.
(29) Schwartz, P. A.; Vetticatt, M. J.; Schramm, V. L. J. Am. Chem.
Soc. 2010, 132, 13425–13433.
This work was supported by NIH grant GM41916. The
authors thank Yong Zhang and Keith Hazleton for their kind
gifts of [3-15N]orotate and UMP synthase, respectively, and Dr.
Andrew S. Murkin and Dr. Luiz Pedro S. de Carvalho, respec-
tively, for insightful discussion about reverse commitment and
for critical reading of this manuscript.
’ REFERENCES
(1) Gao, G.; Nara, T.; Nakajima-Shimada, J.; Aoki, T. J. Mol. Biol.
1999, 285, 149–161.
(30) Prior, J. J.; Santi, D. V. J. Biol. Chem. 1984, 259, 2429–2434.
(31) Renck, D.; Ducati, R. G.; Palma, M. S.; Santos, D. S.; Basso, L. A.
Arch. Biochem. Biophys. 2010, 497, 35–42.
(32) Northrop, D. B. Annu. Rev. Biochem. 1981, 50, 103–131.
(33) Cleland, W. W. Methods Enzymol. 1982, 87, 625–641.
(34) Schramm, V. L. Methods Enzymol. 1999, 308, 301–355.
(35) Murkin, A. S.; Birck, M. R.; Rinaldo-Matthis, A.; Shi, W.;
Taylor, E. A.; Almo, S. C.; Schramm, V. L. Biochemistry 2007,
46, 5038–5049.
(36) Luo, M.; Li, L.; Schramm, V. L. Biochemistry 2008, 47,
2565–2576.
(37) Li, L.; Luo, M.; Ghanem, M.; Taylor, E. A.; Schramm, V. L.
Biochemistry 2008, 47, 2577–2583.
(2) Miller, R. L.; Sabourin, C. L.; Krenitsky, T. A.; Berens, R. L.;
Marr, J. J. J. Biol. Chem. 1984, 259, 5073–5077.
(3) Larson, E. T.; Mudeppa, D. G.; Gillespie, J. R.; Mueller, N.;
Napuli, A. J.; Arif, J. A.; Ross, J.; Arakaki, T. L.; Lauricella, A.; Detitta, G.;
Luft, J.; Zucker, F.; Verlinde, C. L.; Fan, E.; Van Voorhis, W. C.; Buckner,
F. S.; Rathod, P. K.; Hol, W. G.; Merritt, E. A. J. Mol. Biol. 2010,
396, 1244–1259.
(4) Paege, L. M.; Schlenk, F. Arch. Biochem. Biophys. 1952, 40,
42–49.
(5) Pizzorno, G.; Cao, D.; Leffert, J. J.; Russell, R. L.; Zhang, D.;
Handschumacher, R. E. Biochim. Biophys. Acta 2002, 1587, 133–144.
(6) Vita, A.; Huang, C. Y.; Magni, G. Arch. Biochem. Biophys. 1983,
226, 687–692.
(7) Pugmire, M. J.; Ealick, S. E. Biochem. J. 2002, 361, 1–25.
(8) Westheimer, F. H. Chem Rev 1961, 61, 265–273.
(9) Sims, L. B.; Fry, A.; Netherton, L. T.; Wilson, J. C.; Reppond,
K. D.; Crook, S. W. J. Am. Chem. Soc. 1972, 94, 1364–1365.
(10) McCann, J. A.; Berti, P. J. J. Am. Chem. Soc. 2007, 129,
7055–7064.
(38) Cleland, W. W. Arch. Biochem. Biophys. 2005, 433, 2–12.
(39) Northrop, D. B. Biochemistry 1975, 14, 2644–2651.
(40) For this reaction, the computationally predicted 15Keq = 1.007.
Rearranging eq 7 to solve for 15k, one obtains 15k = 15(V/K) ꢁ [1 þ Cf] ꢀ
15
Cf þ [15(V/K) ꢀ Keq] ꢁ Cr, where the terms containing the cor-
rections for Cf and Cr are explicitly separated. Substituting in the values
found in this work, 15k = 1.024 ꢁ [1 þ 0.283] ꢀ 0.283 þ [1.024 ꢀ
1.007] ꢁ Cr, which is then further reduced to 15k = 1.030 þ 0.017 ꢁ Cr.
Because the maximum calculated 1-15N KIE on the arsenolysis of uridine
equals 15k, within experimental error, following correction for Cf, and Cr
cannot assume negative values, it is clear that the only possible value for
Cr is 0.
(11) Kline, P. C.; Schramm, V. L. Biochemistry 1993, 32, 13212–13219.
(12) Singh, V.; Schramm, V. L. J. Am. Chem. Soc. 2006, 128,
14691–14696.
(41) Guthrie, R. D.; Jencks, W. P. Acc. Chem. Res. 1989, 22, 343–349.
(42) Berti, P. J.; McCann, J. A. Chem Rev 2006, 106, 506–555.
(43) Berti, P. J.; Blanke, S. R.; Schramm, V. L. J. Am. Chem. Soc. 1997,
119, 12079–12088.
(13) Berti, P. J.; Schramm, V. L. J. Am. Chem. Soc. 1997, 119,
12069–12078.
(14) Zhang, Y.; Luo, M.; Schramm, V. L. J. Am. Chem. Soc. 2009,
131, 4685–4694.
(44) Hehre, W. J. Acc. Chem. Res. 1975, 8, 369–376.
(45) Cook, P. F.; Cleland, W. W. Biochemistry 1981, 20, 1790–1796.
(46) Lewandowicz, A.; Schramm, V. L. Biochemistry 2004, 43,
1458–1468.
(47) Silva, R. G.; Hirschi, J. S.; Ghanem, M.; Murkin, A. S.; Schramm,
V. L. Biochemistry 2011, 50, 2701–2709.
(48) Werner, R. M.; Stivers, J. T. Biochemistry 2000, 39, 14054–14064.
(49) Komissarov, A. A.; Moltchan, O. K.; Romanova, D. V.; Debabov,
V. G. FEBS Lett. 1994, 355, 192–194.
(15) Schwartz, P. A.; Vetticatt, M. J.; Schramm, V. L. Biochemistry
2011, 50, 1412–1420.
(16) Cen, Y.; Sauve, A. A. J. Am. Chem. Soc. 2010, 132, 12286–12298.
(17) Miles, R. W.; Tyler, P. C.; Furneaux, R. H.; Bagdassarian, C. K.;
Schramm, V. L. Biochemistry 1998, 37, 8615–8621.
(18) Singh, V.; Shi, W.; Evans, G. B.; Tyler, P. C.; Furneaux, R. H.;
Almo, S. C.; Schramm, V. L. Biochemistry 2004, 43, 9–18.
(19) Singh, V.; Lee, J. E.; Nunez, S.; Howell, P. L.; Schramm, V. L.
Biochemistry 2005, 44, 11647–11659.
(50) Fedorov, A.; Shi, W.; Kicska, G.; Fedorov, E.; Tyler, P. C.;
Furneaux, R. H.; Hanson, J. C.; Gainsford, G. J.; Larese, J. Z.; Schramm,
V. L.; Almo, S. C. Biochemistry 2001, 40, 853–860.
(51) Pauling, L. J. Am. Chem. Soc. 1947, 69, 542–553.
(52) Houk, K. N.; Gustafson, S. M.; Black, K. A. J. Am. Chem. Soc.
1992, 114, 8565–8572.
(53) Schramm, V. L. Annu. Rev. Biochem. 1998, 67, 693–720.
(54) Zhang, Y.; Schramm, V. L. J. Am. Chem. Soc. 2010, 132,
8787–8794.
(55) Berti, P. J.; Tanaka, K. S. E. Adv. Phys. Org. Chem. 2002, 37,
239–314.
(56) Chen, X. Y.; Berti, P. J.; Schramm, V. L. J. Am. Chem. Soc. 2000,
122, 6527–6534.
(20) Parkin, D. W.; Leung, H. B.; Schramm, V. L. J. Biol. Chem. 1984,
259, 9411–9417.
(21) Laemmli, U. K. Nature 1970, 227, 680–685.
(22) Miller, R. L.; Lindstead, D. Mol. Biochem. Parasitol. 1983,
7, 41–51.
(23) Rose, I. A. Methods Enzymol. 1980, 64, 47–59.
(24) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.;
Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.;
Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;
Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima,
T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.;
Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K.
N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.;
Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.;
Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.;
Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.;
Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.;
Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich,
(57) Gawlita, E.; Szylhabel-Godala, A.; Paneth, P. J. Phys. Org. Chem.
1996, 9, 41–49.
€
S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.;
9931
dx.doi.org/10.1021/ja2031294 |J. Am. Chem. Soc. 2011, 133, 9923–9931