The kinetic mechanism of human uridine phosphorylase 1: Towards the development of enzyme inhibitors for cancer chemotherapy

Article abstract of DOI:10.1016/j.abb.2010.03.004

Uridine phosphorylase (UP) is a key enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate (R1P). The human UP type 1 (hUP1) is a molecular target for the design of inhibitors intended to boost endogenous uridine levels to rescue normal tissues from the toxicity of fluoropyrimidine nucleoside chemotherapeutic agents, such as capecitabine and 5-fluorouracil. Here, we describe a method to obtain homogeneous recombinant hUP1, and present initial velocity, product inhibition, and equilibrium binding data. These results suggest that hUP1 catalyzes uridine phosphorolysis by a steady-state ordered bi bi kinetic mechanism, in which inorganic phosphate binds first followed by the binding of uridine, and uracil dissociates first, followed by R1P release. Fluorescence titration at equilibrium showed cooperative binding of either P_i or R1P binding to hUP1. Amino acid residues involved in either catalysis or substrate binding were proposed based on pH-rate profiles.

Full text of DOI:10.1016/j.abb.2010.03.004

Archives of Biochemistry and Biophysics 497 (2010) 35–42
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics
journal homepage: www.elsevier.com/locate/yabbi
The kinetic mechanism of human uridine phosphorylase 1: Towards the
development of enzyme inhibitors for cancer chemotherapy q
a,b,
Daiana Renck a,b, Rodrigo G. Ducati a, Mario S. Palma c, Diógenes S. Santos a,b, , Luiz A. Basso
*
**
a Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB),
Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), 6681/92-A Av. Ipiranga, 90619-900 Porto Alegre, RS, Brazil
b Programa de Pós-Graduação em Biologia Celular e Molecular, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil
c Laboratório de Biologia Estrutural e Zooquímica, Centro de Estudos de Insetos Sociais, Departamento de Biologia, Instituto de Biociências de Rio Claro,
Universidade Estadual Paulista (UNESP), Rio Claro, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Uridine phosphorylase (UP) is a key enzyme in the pyrimidine salvage pathway, catalyzing the reversible
phosphorolysis of uridine to uracil and ribose-1-phosphate (R1P). The human UP type 1 (hUP1) is a molec-
ular target for the design of inhibitors intended to boost endogenous uridine levels to rescue normal tissues
from the toxicity of fluoropyrimidine nucleoside chemotherapeutic agents, such as capecitabine and 5-flu-
orouracil. Here, we describe a method to obtain homogeneous recombinant hUP1, and present initial veloc-
ity, product inhibition, and equilibrium binding data. These results suggest that hUP1 catalyzes uridine
phosphorolysis by a steady-state ordered bi bi kinetic mechanism, in which inorganic phosphate binds first
followed by the binding of uridine, and uracil dissociates first, followed by R1P release. Fluorescence titra-
tion at equilibrium showed cooperative binding of either Pi or R1P binding to hUP1. Amino acid residues
involved in either catalysis or substrate binding were proposed based on pH-rate profiles.
Ó 2010 Elsevier Inc. All rights reserved.
Received 18 January 2010
and in revised form 5 March 2010
Available online 11 March 2010
Keywords:
Cancer chemotherapy
Initial velocity
Product inhibition
Fluorescence spectroscopy
pH-rate profiles
Uridine phosphorylase kinetic mechanism
Pyrimidine nucleoside phosphorylases are key enzymes in the
pyrimidine salvage pathway. Two types of enzymes have been
identified in human cells: uridine phosphorylase (UP1; EC 2.4.2.3)
and thymidine phosphorylase (EC 2.4.2.4) [1–3]. UP belongs to the
nucleoside phosphorylase (NP) super-family of proteins, in the NP-
1 subset [4], and plays an important role in nucleoside metabolism,
catalyzing the phosphorolysis of uridine (Urd) to uracil and ribose-1-
phosphate (R1P) (Fig. 1). These products can be further utilized for
nucleoside synthesis [5,6]. In humans, there are two isoforms of
UP, hUP1 [3] and hUP2 [2], which are encoded by two different genes
in distinct chromosomes. The alignment of the isoforms showed that
they share approximately 60% amino acid sequence identity [2]. The
hUP1 cDNA contains an open reading frame coding for a sequence of
310 amino acid residues corresponding to a protein with a subunit
molecular mass of 33.9 kDa that is dimeric in solution [3,7], which
is in contrast to the hexameric Escherichia coli enzyme [8,9].
Although UP is present in most normal and tumoral tissues, its
activity as well as its expression is elevated in certain tumors, a
feature that may contribute to selectivity of chemotherapeutic
agents [3,10–12]. Studies have shown that this expression can be
up-regulated by treating tumor cells with cytokines such as inter-
feron-a, interferon-c, tumor necrosis factor-a, and interleukin-1a
[3,13]. RT-PCR analysis demonstrated that basic fibroblast growth
factor (bFGF) increases the expression level of mouse UP1 in oste-
olineage cell lines; the induction by bFGF is dependent of NFjB
q
This work was supported by the National Institute of Science and Technology on
Tuberculosis (DECIT/SCTIE/MS-MCT-CNPq-FNDCT-CAPES) and the Millennium Ini-
tiative Program (CNPq) to D.S.S. and L.A.B. D.S.S. (CNPq, 304051/1975-06), L.A.B.
(CNPq, 520182/99-5), and M.S.P. (CNPq, 500079/90-0) are Research Career Awar-
dees of the National Research Council of Brazil (CNPq). R.G.D. is a post-doctoral
fellow of CNPq. D.R. is recipient of an MSc scholarship awarded by BNDES.
* Corresponding author. Fax: +55 51 33203629.
** Corresponding author at: Centro de Pesquisas em Biologia Molecular
e
Funcional (CPBMF), Instituto Nacional de Ciência e Tecnologia em Tuberculose
(INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), 6681/92-
A Av. Ipiranga, 90619-900 Porto Alegre, RS, Brazil. Fax: +55 51 33203629.
E-mail addresses: diogenes@pucrs.br (D.S. Santos), luiz.basso@pucrs.br (L.A.
Basso).
1
Abbreviations used: Arg, arginine; BAU, 5-benzylacyclouridine; CV, column volume;
bFGF, basic fibroblast growth factor; ESI-MS, electrospray ionization mass spectrom-
etry; EWS, Ewing’s sarcomas; 5-FU, 5-fluorouracil; FUrd, fluorouridine; FUMP, fluoro-
uridine monophosphate; FUDP, fluorouridine diphosphate; FUTP, fluorouridine
triphosphate; FdUrd, fluorodeoxyuridine; FdUMP, fluorodeoxyuridine monophos-
phate; FdUDP, fluorodeoxyuridine diphosphate; FdUTP, fluorodeoxyuridine triphos-
phate; Hepes, N-2-hydroxyethylpiperazyne-N0-2-ethanesulfonic acid; His, histidine;
hUP1, human uridine phosphorylase 1; IPTG, isopropyl-b-D-thiogalactopyranoside; LB,
Luria–Bertani; NP, nucleoside phosphorylase; OPRT, orotate phosphoribosyltransfer-
ase; Pi, inorganic phosphate; PNP, purine nucleoside phosphorylase; R1P, ribose-1-
phosphate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel eletrophoresis; TB,
terrific broth; TK, thymidine kinase; TP, thymidine phosphorylase; Tris, tris(hydroxy-
methyl)aminomethane; Urd, uridine; UK, uridine kinase; UP, uridine phosphorylase.
activity [14]. In addition, it has been shown that tumor-associated
chromosomal translocation in Ewing’s family tumors, where the
N-terminus of Ewing’s sarcoma (EWS) gene fuses with the C-termi-
nus of some ETS transcription factors, up-regulates UP promoter
0003-9861/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.abb.2010.03.004

36
D. Renck et al. / Archives of Biochemistry and Biophysics 497 (2010) 35–42
Fig. 1. Chemical reaction catalyzed by hUP1.
in vivo, suggesting that UP could be a direct target of EWS/ETS
fusion proteins [15,16]. In contrast, the activity and gene promoter
levels of UP have been shown to be down-regulated by wild-type
p53, a tumor suppressor gene that plays a key role in cell growth
control, DNA damage repair, and apoptosis [1,17].
to rescue 5-FU toxicity. Acyclouridine analogues, including 5-ben-
zylacyclouridine (BAU), have been designed as hUP1 inhibitors,
and BAU has been shown in clinical trials to be capable of increas-
ing plasma Urd concentration, thereby increasing the therapeutic
index of 5-FU [7].
UP plays an important role in the homeostatic regulation of Urd
concentration in plasma (1–5 lM) and tissues, and affects activa-
Enzyme inhibitors make up roughly 25% of the drugs marketed in
United States [21]. Enzymes catalyze multistep chemical reactions
to achieve rate accelerations by stabilization of the transition-state
structure [22]. Accordingly, mechanistic analysis (kinetic, chemical,
and catalytic mechanisms) should always be a top priority for en-
zyme-targeted drug programs aiming at the rational design of po-
tent enzyme inhibitors. Here we describe amplification, cloning,
and sequencing of the recombinant hUP1 coding gene. We also pres-
ent heterologous protein expression in E. coli, purification to homo-
geneity, N-terminal amino acid sequencing, electrospray ionization
mass spectrometry analysis, determination of true steady-state
kinetic parameters, product inhibition, equilibrium fluorescence of
substrate/product binding, and pH-rateprofiles of functional recom-
binant hUP1 enzyme. These results provide a solid foundation on
which to base function-guided hUP1 enzyme inhibitors with poten-
tial anti-cancer activity.
tion and catabolism of several nucleoside analogues used in cancer
chemotherapy [2]. These analogues include fluoropyrimidines,
such as 5-fluorouracil (5-FU) [1,8], which is a uracil analogue with
a fluorine atom at the C-5 position. This compound was developed
in 1957 [18] after the observation that rat tumoral tissues use ura-
cil more rapidly than normal tissues, which indicated that uracil
metabolism is a potential target for chemotherapy [8]. Since then,
5-FU has been used in clinical practice against many types of solid
tumors; yet, most of its use is related to colorectal cancer [8,19]. 5-
FU is converted to fluorodeoxyuridine monophosphate (FdUMP),
fluorodeoxyuridine triphosphate (FdUTP), and fluorouridine tri-
phosphate (FUTP), the three main active metabolites. The main
mechanism of 5-FU activation is either directly by orotate phos-
phoribosyltransferase (OPRT), or indirectly by the sequential action
of UP converting 5-FU to fluorouridine (FUrd) which, in turn, is
converted to fluorouridine monophosphate (FUMP) by uridine ki-
nase (UK) [8,14]. FUMP is phosphorylated to fluorouridine diphos-
phate (FUDP), which can be either phosphorylated to FUTP or
converted by ribonucleotide reductase into fluorodeoxyuridine
diphosphate (FdUDP) [8]. The latter, in turn, can either be phos-
phorylated to FdUTP or dephosphorylated to FdUMP, both of which
are active metabolites. There is also an alternative pathway that in-
volves conversion of 5-FU to fluorodeoxyuridine (FdUrd) by thymi-
dine phosphorylase (TP), and conversion of FdUrd to FdUMP by
thymidine kinase (TK). The main metabolites of 5-FU exert their
anti-cancer activity either by disrupting RNA synthesis (FUTP), or
inhibiting thymidylate synthase (TS) enzyme activity (FdUMP) that
converts dUMP to deoxythymidine monophosphate (dTMP), which
is needed for DNA synthesis and repair. In addition, inhibition of TS
by FdUMP results in accumulation of dUMP leading to increased
levels of dUTP. The latter and the 5-FU metabolite FdUMP can be
misincorporated into DNA. Clinical evidences have demonstrated
the ability of Urd to reduce bone marrow and gastrointestinal tox-
icity induced by 5-FU, called ‘‘rescue of 5-FU toxicity” [2,6,10,20].
High doses of uridine are required to produce the ‘‘rescue” effect
due to the short half-life of plasma uridine of only 2 min and the
Materials and methods
Amplification and cloning of the human UPP1 gene
The human UPP1 coding sequence was searched on the Gene-
Bank (BC007348) of the National Institute for Biotechnology Infor-
mation (http://www.ncbi.nlm.nhi.gov).
The cDNA of UPP1 was obtained by RT-PCR amplification of
colorectal RNA (from Ambion; Austin, TX, USA). The oligonucleo-
tide primers used (forward primer, 50-CAGTTGGCCATATGGCGGC
CACGGGAGC-30; and reverse primer, 50-GCGGAGAAGCTTGGCAGC
GCTCAGGCC-30) contained, respectively, NdeI and HindIII (New
England Biolabs) restriction sites (underlined). The PCR product
was analyzed on 1% agarose gel, and a 930-bp band was detected
and purified. The DNA fragment was cloned into pCR-Blunt cloning
vector (Invitrogen), cleaved with NdeI and HindIII restriction en-
zymes, and subcloned into the pET-23a(+) expression vector
(Novagen). The complete UPP1 gene sequence was determined by
automated DNA sequencing to confirm sequence integrity and
the absence of mutations in the cloned fragment.
regulation of plasma uridine homeostasis at the 2–4
lM level by
the activity of hepatic UP [6,10]. However, these high doses needed
to produce this effect are not well tolerated and produce dose-lim-
iting effect in humans. It is thus necessary to develop a drug capa-
ble of maintaining elevated endogenous levels of Urd [6,7,10] to
protect the normal tissues through Urd rescue effect. Accordingly,
inhibition of UP activity appears an attractive therapeutic strategy
Expression and purification of recombinant hUP1
The pET-23a(+)::UPP1 recombinant plasmid was transformed
into E. coli Rosetta (DE3) competent cells (Novagen) and selected
on Luria–Bertani (LB) agar plates containing 50
l
g mLꢀ1 ampicillin
and 34
l
g mLꢀ1 cloranfenicol. A single colony was grown overnight

D. Renck et al. / Archives of Biochemistry and Biophysics 497 (2010) 35–42
37
in LB medium pH 7.2 (60 mL) containing the same antibiotics, at
37 °C. An aliquot of this culture (10 mL) was used to inoculate ter-
rific broth (TB) medium (2.5 L, with the same antibiotics) and
grown for 36 h at 30 °C after reaching an OD600 nm of 0.4–0.6, with-
performed under initial rate conditions at 37 °C and 100 mM Tris
pH 7.4, in 500 L total reaction volumes for 60 s. This assay was
based on the maximum difference in absorbance at 280 nm be-
tween Urd and uracil (
A = 2100 Mꢀ1 cmꢀ1), in which a decrease
l
D
out isopropyl-b-
D
-thiogalactopyranoside (IPTG) induction. The
in absorbance is observed upon conversion of Urd to uracil [27].
One unit (U) of enzyme activity was defined as the amount of en-
zyme that catalyses the formation of 1 lmol of product per min.
same procedure was employed for E. coli Rosetta (DE3) cells trans-
formed with pET-23a(+) (control). The cells (40 g) were harvested
by centrifugation at 11,800g for 30 min at 4 °C and stored at
ꢀ20 °C. Soluble protein expression was analyzed by 12% sodium
dodecyl sulfate–polyacrylamide gel eletrophoresis (SDS–PAGE)
stained with Coomassie Brilliant Blue [23].
Initial velocity measurements
Initial velocity studies were carried out to determine the true
steady-state kinetic parameters, in the forward direction. Satura-
tion curves were performed varying concentrations of Urd (20–
500 lM) against several fixed-varying concentrations of inorganic
phosphate (Pi) (1–10 mM).
All purification steps were performed at 4 °C and sample elution
was monitored by UV detection. Frozen cells (5 g) were suspended
in 25 mL of 50 mM N-2-hydroxyethylpiperazyne-N0-2-ethanesul-
fonic acid (Hepes) pH 7.0 (buffer A) and incubated with
0.2 mg mLꢀ1 lysozyme (Sigma) for 30 min. The cells were dis-
rupted by sonication (five pulses of 10 s) and the solution was
cleared by centrifugation at 48,000g for 30 min. The supernatant
was treated with 1% (wt/vol) streptomycin sulfate (Sigma; final
concentration) for 30 min to precipitate the nucleic acids, and cen-
trifuged (48,000g for 30 min). The supernatant was dialyzed
against buffer A (2 ꢁ 2 L, 3 h each). Residual precipitate was re-
moved by centrifugation (48,000g for 30 min) and the supernatant
was loaded onto an SP Sepharose Fast Flow cation exchange col-
umn (GE Healthcare) pre-equilibrated with buffer A. The column
was washed with five column volumes (CV) of buffer A and the ad-
sorbed material was eluted with 15 CV linear gradient (0–100%) of
50 mM Hepes 200 mM NaCl pH 7.0 (buffer B) at a 1 mL minꢀ1 flow
rate. The target recombinant protein was eluted at approximately
half of the gradient, where a single SDS–PAGE band could be ob-
served. The homogenous recombinant protein was dialyzed
against 100 mM tris(Hydroxymethyl)aminomethane (Tris) pH 7.4
(3 ꢁ 2 L, 3 h each), concentrated, and stored at ꢀ80 °C. Protein con-
centration was determined with Bradford Protein Assay Kit (Bio-
Rad Laboratories) and bovine serum albumin was used as standard
[24]. All subsequent activity and binding assays were performed in
100 Tris pH 7.4, unless stated otherwise.
Product inhibition patterns
To provide an additional experimental approach to distinguish
between the possible kinetic mechanisms, product inhibition stud-
ies were carried out at varying concentrations of one substrate, fixed
concentrations of the co-substrate (in nonsaturating levels), and
fixed-varying concentrations of products (either R1P or uracil). The
experimental conditions were as follows: varying Urd concentra-
tions (20–500
concentrations of either uracil (50–600
varying Pi concentrations (1–10 mM), fixed Urd concentration
lM), fixed Pi concentration (2 mM), and fixed-varying
l
M) or R1P (24–160 M);
l
(50
300
l
l
M), and fixed-varying concentrations of either uracil (50–
M) or R1P (80–320 M).
l
Equilibrium binding by fluorescence spectroscopy
Fluorescence measurements were carried out in an RF-5301 PC
Spectrofluorophotometer (Shimadzu) at 25 °C. Measurements of
intrinsic hUP1 protein fluorescence employed excitation wave-
length at 280 nm in each binding experiment, and the emission
wavelength ranged from 285 to 350 nm. The slits for excitation
and emissionwere both3 nm. Fluorescencetitrations of binarycom-
plex formation were carried out by making microliter additions of
Amino acid sequence and mass spectrometry analysis
the following compounds to 2 mL containing 10
Pi stock solution (19.99–434.2 M final concentration); 5 mM Urd
stock solution (2.498–97.92 M final concentration); 10 mM uracil
stock solution (4.997–123.3
stock solution (1.99–41.53
ments were employed to both determine the maximum ligand con-
centrations to be used with no inner filter effect and to account for
any dilution effect on protein fluorescence.
lM hUP1: 40 mM
The N-terminal amino acid sequence of homogeneous recombi-
nant hUP1 protein was analyzed by automated Edman degradation
sequencingusingagas-phasesequencerPPSQ-21A(Shimadzu)[25].
hUP1 was assessed by electrospray ionization mass spectrometry
(ESI-MS) according to Chassaigne and Lobinski, with some adapta-
tions [26]. The sample was analyzed on Quattro-II triple-quadrupole
mass spectrometer (Micromass; Altrincham, UK). During all experi-
ments, the source temperature was maintained at ꢀ80 °C and the
l
l
l
l
M final concentration); 4 mM R1P
M final concentration). Control experi-
capillary voltage at 3.6 kV; a drying nitrogen gas flow (200 L hꢀ1
)
pH-rate profiles
and a nebulizer gas flow (20 L hꢀ1) were used. Intact horse heart
myoglobin was used to calibrate the mass spectrometer and its typ-
ical cone voltage-induced fragments. hUP1 subunit molecular mass
was determined by adjusting the mass spectrometer to yield a peak
with a half-height of one mass unit, and the sampling cone-to-skim-
mer lens voltage controlling the transfer of ions to the mass analyzer
was set to 38 V. Approximately 50 pmol of each sample were in-
jected into the electrospray transport solvent. The ESI spectrum
was obtained in the multichannel acquisition mode, with scanning
from 500 to 1,800 m/z at a scan time of 7 s. The mass spectrometer
is equipped with MassLynx and Transform softwares for data acqui-
sition and spectrum handling.
To determine the dependence of the kinetic parameters on pH,
initial velocities were measured in the presence of varying concen-
trations of one substrate and a saturating level of the other in a buffer
mixture of 2-(N-morpholino)ethanesulfonic acid /Hepes/2-(N-
cyclohexylamino)ethanesulfonic acid over the following pH values:
5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0 [28]. The data were plotted
as pH values versus either log kcat or log kcat/KM. As the KM values
changed as a function of pH, different concentration ranges of the
variable substrate as well as the fixed substrate had to be employed.
ForvaryingUrdconcentrationstheexperimentalconditionswere:at
pH 5.0: Pi = 20 mM, 200
l
M 6 Urd 6 900
M; at pH 6.0: Pi = 12 mM, 20
M; at pH 6.5: Pi = 10 mM, 20 M 6 Urd 6 700 M; at pH 7.0
M 6 Urd 6 500 M; at pH 8.0: Pi = 10 mM,
M; at pH 8.5: Pi = 10 mM, 20 M 6 Urd 6
M; and at pH 9.0: Pi = 10 mM, 20 M 6 Urd 6 800 M. For
l
M; at pH 5.5: Pi = 20 mM,
100
800
l
l
M 6 Urd 6 800
l
lM 6 Urd 6
hUP1 enzymatic assay
l
l
and 7.5: Pi = 10 mM, 20
20 M 6 Urd 6 700
600
l
l
Recombinant hUP1 enzyme activity was monitored in an UV-
2550 UV/Visible spectrophotometer (Shimadzu). All assays were
l
l
l
l
l
l

38
D. Renck et al. / Archives of Biochemistry and Biophysics 497 (2010) 35–42
varying Pi concentrations the experimental ranges employed were
as follows: at pH 5.0: Urd = 800 M, 2 mM 6 Pi 6 20 mM; at pH
5.5: Urd = 700 M, 2 mM 6 Pi 6 16 mM; at pH 6.0: Urd = 700 M,
0.5 mM 6 Pi 6 8 mM; at pH 6.5: Urd = 600 M, 0.05 mM 6 Pi 6
8 mM; at pH 7.0 and 7.5: Urd = 500 M, 0.1 mM 6 Pi 6 8 mM; at
pH 8.0: Urd = 600 M, 0.1 mM 6 Pi 6 8 mM; at pH 8.5: Urd =
500 M,
and low pH are required to achieve high-level expression in the ab-
sence of IPTG induction, which may be part of a general cellular re-
sponse to nutrition limitation [32]. However, more recently, it has
been shown that unintended induction in the pET system is due to
the presence of as little as 0.0001% of lactose in the medium [33]. It
is noteworthy that a large amount of recombinant hUP1 protein re-
mained in the insoluble fraction (Fig. 2, lane 3). Although inclusion
body formation can greatly simplify protein purification, there is
no guarantee that the in vitro refolding will yield large amounts
of biologically active protein. Moreover, inclusion body purification
schemes present a number of problems such as: use of denaturants
that are expensive and can cause irreversible modifications of pro-
tein structure that will elude all of the most sophisticated analyti-
cal tests, refolding usually must be done in very dilute solution and
the protein reconcentrated, and refolding encourages protein
isomerization leading to precipitation during storage [34]. Since
we aimed at determining the mode of action of recombinant
hUP1 enzyme, we deemed more appropriate to avoid solubilizing
agents.
l
l
l
l
l
l
l
M, 0.05 mM 6 Pi 6 5 mM; and at pH 9.0: Urd = 700 l
0.05 mM 6 Pi 6 5 mM.
Results and discussion
Amplification, cloning, and DNA sequencing
The PCR amplification protocol yielded a product with an ex-
pected size corresponding to the human UPP1 (930-bp) DNA cod-
ing sequence (data not shown). The fragment was purified from
the agarose gel and ligated into the pET-23a(+) expression vector.
Nucleotide sequence analysis confirmed both identity and integ-
rity of human UPP1 DNA coding sequence.
Purification of recombinant hUP1 protein
Expression of recombinant hUP1 protein
Recombinant hUP1 protein was efficiently purified to homoge-
neity (Fig. 3) by a single-step purification protocol, using a cation
exchange column. The target protein eluted at approximately
50% of buffer B. The 3.8-fold purification protocol resulted in a pro-
tein yield of 43% and 20.8 mg of active recombinant hUP1 protein
from 5 g of cells (Table 1). In contrast to Roosild et al. [7], we found
no need to add potassium salt in the purification buffers to prevent
protein aggregation and precipitation. The homogeneous protein
was stored at ꢀ80 °C, with no loss of activity.
The pET-23a(+)::UPP1 recombinant plasmid was transformed
into E. coli Rosetta (DE3) host cells by electroporation. Analysis
by SDS–PAGE (Fig. 2) showed that the cell extracts contained the
recombinant protein, in the insoluble and soluble fraction, with
an apparent molecular mass of 33 kDa, in agreement with the ex-
pected size of 33.934 kDa for hUP1. Among a number of protocols
tested, the best experimental condition for expression of recombi-
nant hUP1 occurred with E. coli Rosetta (DE3) cells grown for 36 h
(after reaching an OD600 nm of 0.4–0.6, without IPTG induction) at
30 °C in TB medium. The pET expression vector system (Novagen)
has a strong IPTG-inducible bacteriophage T7 lacUV5 late promoter
that controls the T7 RNA polymerase to transcribe cloned target
genes [29]. It has been shown that lac-controlled systems could
have high-levels of protein expression in the absence of inducer
[30,31]. It has been proposed that leaky protein expression is due
to derepression of the lac-controlled system when cells approach
stationary phase in complex medium and that cyclic AMP, acetate,
Mass spectrometry and N-terminal amino acid sequencing
The subunit molecular mass value for hUP1 was determined to
be 33,934.00 Da by electrospray ionization mass spectrometry
(ESI-MS). Since the predicted molecular mass is 33,934.00 Da, this
result indicates removal of the N-terminal methionine (predicted
methionine molecular mass = 131.20 Da). These results provide
evidence that confirm the identity of recombinant hUP1.
The Edman degradation method identified the first 21 N-termi-
nal amino acid residues of the recombinant hUP1 as: AATGANAE-
KAESHNDCPVRLL. This result unambiguously demonstrates that
the purified protein is hUP1, and confirms the removal of the
N-terminal methionine. Protein N-terminal methionine excision
is a common type of post-translational modification process that
occurs in the cytoplasm of many organisms displaying protein syn-
thesis. The cleavage of the initiator methionine is usually directed
by the penultimate amino acid residues with the smallest side
chain radii of gyration (Gly, Ala, Ser, Thr, Pro, Val, and Cys) [35],
which is in agreement with removal of the N-terminal methionine
Fig. 2. The 12% SDS–PAGE analysis of total insoluble and soluble proteins.
Expression of hUP1 after 36 h of cell growth after reaching an OD600 nm of 0.4–0.6
in TB medium without addition of IPTG. Lane 1, Protein Molecular Weight Marker
(Fermentas); lane 2, insoluble E. coli Rosetta (DE3) [pET-23a(+) (control)] extract;
lane 3, insoluble E. coli Rosetta (DE3) [pET-23a(+)::UPP1] extract; lane 4, soluble
E. coli Rosetta (DE3) [pET-23a(+) (control)] extract; lane 5, soluble E. coli Rosetta
(DE3) [pET-23a(+)::UPP1] extract.
Fig. 3. The 12% SDS–PAGE analysis of pooled fractions from hUP1 purification steps.
Lane 1, Protein Molecular Weight Marker (Fermentas); lane 2, crude extract; lane 3,
SP Sepharose Fast Flow cation exchange elution.

D. Renck et al. / Archives of Biochemistry and Biophysics 497 (2010) 35–42
39
Table 1
Product inhibition
Purification of hUP1 from E. coli Rosetta (DE3). Typical purification protocol from 5 g
wet cell paste (300 mL of culture).
The initial velocity results described above cannot distinguish
between a steady-state ordered bi bi mechanism and rapid equilib-
rium random bi bi system. Accordingly, product (either R1P or ura-
cil) inhibition measurements were carried out to determine the
order of substrate addition to the enzyme. The data were fitted
to an equation for either competitive or noncompetitive inhibition:
Purification
step
Total
protein enzyme
(mg)
Total
Specific Purification Yield (%)
activity fold
activity (U) (U mgꢀ1
)
Crude extract
180.3 367.5
2.04
7.70
1.0
3.8
100
43
SP Sepharose Fast Flow 20.8 159.9
v
= VA/[Ka(1 + I/Kis) + A] or v = VA/[Ka(1 + I/Kis) + A(1 + Kii)], respec-
tively. The double-reciprocal plots revealed a pattern of three non-
competitive and one competitive inhibition (Table 2). This pattern
is in agreement with a steady-state ordered bi bi kinetic mecha-
nism [36], in which Pi binds first to free hUP1 enzyme followed
by Urd binding to form the catalytically competent ternary com-
plex. This pattern also suggests that uracil is released first followed
by R1P dissociation from the R1P–hUP1 binary complex. In addi-
tion, a steady-state random bi bi mechanism could be discarded
because double-reciprocal plots were linear in initial velocity stud-
ies and the pattern of product inhibition would be noncompetitive
for all substrate–product pairs. The steady-state ordered bi bi ki-
netic mechanism for hUP1 is in agreement with the cytoplasmic
rat liver UP [19,37]. However, this mechanism is in disagreement
with the random order of substrate addition for UP from both
E. coli [38,39] and Lactobacillus casei [40], and in disagreement with
the ordered mechanism for UP from guinea pig, in which binding of
uracil precedes that of Pi [41].
from hUP1 since alanine is the penultimate N-terminal amino acid
residue.
Initial velocity and steady-state kinetic parameters
The double-reciprocal plots for the forward reaction (phospho-
rolysis) showed a family of lines intersecting to the left of the y-axis
(Fig. 4A and B), which is consistent with ternary complex formation
and a sequential mechanism [36]. Ping-pong and rapid equilibrium
ordered mechanisms could be ruled out, since these mechanisms
display parallel lines and intersecting lines at the y-axis, respec-
tively. The data were fitted to the following equation:
(KiaKb + KaB + KbA + AB), yielding the following true steady-state
v = VAB/
kinetic parameters: kcat = 7.5 ( 0.2) sꢀ1, KUrd = 51 ( 4)
l
M, KP
¼
i
2462ðꢂ228Þ
l
M, kcat/KUrd = 14.7 ( 1.2) ꢁ 104 Mꢀ1 sꢀ1
, and kcat=
KP ¼ 3:05ðꢂ0:28Þ ꢁ 103 Mꢀ1 sꢀ1. These values are different from
i
the apparent steady-state kinetic constants reported for human liver
UP1 [10]. Although it is not possible to offer a clear explanation for
these differences, here we present the true steady-state kinetic
parameters whereas Liu et al. [10] presented apparent steady-state
kinetic parameters.
Equilibrium binding of ligands to hUP1
Binding experiments were employed to confirm the order of
substrate addition proposed by product inhibition for hUP1, and
to reinforce the proposal of the order of product dissociation from
the catalytically competent ternary complex. Intrinsic hUP1 fluo-
rescence enhanced upon either Pi or R1P binding to free hUP1. Plots
of either Pi or R1P concentration versus relative protein fluores-
cence variation upon binary complex formation (Fig. 5A and B)
were sigmoidal, and the data were fitted to the Hill equation
[42]: F/Fmax = An/(K0 + An), yielding values of K0 = 106 ( 56) mM
and n = 2.0 ( 0.1) for Pi, and K0 = 297 ( 102)
lM and n = 2.0 ( 0.1)
for R1P. K0 represents the mean dissociation constant for hUP1:li-
gand binary complex formation, which is composed of interaction
factors and the intrinsic dissociation constant, and n represents the
total number of binding sites [36]. The value of 2 for n is in agree-
ment with the dimeric form of hUP1 in solution demonstrated by
size-exclusion chromatography and multi-angle static light scat-
tering [7]. Positive cooperativity in the binding of Pi or R1P to
hUP1 was supported by upward-curved double-reciprocal plots
(insets in Fig 5A and B, respectively). No enhancement in intrinsic
protein fluorescence could be detected upon binding of either uri-
dine or uracil to free hUP1, thereby lending support to the pro-
posed kinetic mechanism for hUP1. Interestingly, based on
crystal structures of ternary complexes of E. coli UP either with
Table 2
Product inhibition patterns for hUP1.a
Variable
substrate
Product
inhibitor
Inhibition
typeb
Kis
(l
M)c
Kii (l
M)d
Uridine
Uridine
Pi
Uracil
R1P
Uracil
R1P
NC
NC
NC
C
50
34
137 27
494 61
7
7
618 119
2.16 ꢁ 106e
251 20
–
Pi
a
b
c
At 37 °C and 100 mM Tris pH 7.4.
NC = noncompetitive, C = competitive.
Kis is the slope inhibition constant.
Kii is the intercept inhibition constant.
Fig. 4. Intersecting initial velocity patterns for hUP1 with either Urd (A) or Pi (B) as
the variable substrate. Each curve represents fixed-varying levels of the co-
substrate.
d
e
This value was poorly defined due to a large SE value.

40
D. Renck et al. / Archives of Biochemistry and Biophysics 497 (2010) 35–42
tion of a single ionizable group in each limb. The data from pH 5.0–
6.0 for kcat=KP , and pH 5.0 for kcat/KUrd were not included in the
i
analysis, since these saturation curves were sigmoidal. It is inter-
esting to note that, as has been pointed out in a recent review
showing a timeline of evolution of allostery as a concept [43], pH
is now considered an allosteric effector. At any rate, as the intracel-
lular pH is near neutral, we deemed more appropriate to consider
only the pH values that yielded hyperbolic curves to allow a more
straightforward data analysis.
The pH-rate profile for kcat indicates that protonation of a group
with pKa value of 5.5 ( 0.6) and deprotonation of another group
with pKa value of 8.2 ( 0.9) play a critical role in hUP1 enzyme
catalysis (Fig. 7A). The amino acid side chain having a pKa value
of 5.5 that has to be deprotonated for efficient catalysis may tenta-
tively be ascribed to the conserved His36 residue showed by
crystallography to interact with the acyclic moiety of 5-benzylacy-
clouridine (BAU, an inhibitor of hUP1) [7]. In the usually predom-
inant nonionized tautomeric form of His36, the N-3 nitrogen (e2)
with the hydrogen atom may act as an electrophile and H-bond do-
nor, whereas N-1 nitrogen (d1) atom may act as a nucleophile and
acceptor for H-bonding. However, the position of the hydrogen
atom can vary with conditions in the local environment of hUP1
active site. The conserved Tyr35 is a candidate for the residue hav-
ing a pKa value of 8.2 that has to be protonated for efficient hUP1
catalysis to occur.
The pH dependence of kcat=KP (Fig. 7B) indicates that proton-
i
ation of a group and deprotonation of another group with an aver-
age pKa value of 7.7 ( 0.8) abolish Pi binding. These data suggest
that there is no pH plateau in which hUP1 would be fully in its ac-
tive form as regards Pi binding. In other words, with increasing pH,
before one group has been fully deprotonated to give its active
form, another group required in the protonated form has started
to lose its proton. Amino acid sequence comparison demonstrates
conservation of three arginine residues (Arg64, Arg94, and Arg138)
in hUP1. Although these residues are involved in Pi binding, the
crystal structure of hUP1 has shown that Arg64 is bent away from
the Pi anion in the active site, leaving two guanidinium groups
(Arg94 and Arg138), one from each subunit of the dimer, to bind
the substrate [7]. Based on this finding, the Arg64 residue has been
suggested not to participate in Pi binding [7]. The pKa value of the
d-guanido group of arginine in solution is usually about 12. How
can one reconcile the pKa values of 7.7 for amino acid side chains
involved in Pi binding? It has been pointed out by Copeland [44]
that in some cases the pKa values that are measured cannot be cor-
rectly ascribed to a particular amino acid, but rather reflect a spe-
cific set of residue interactions within an enzyme molecule that
create in situ a unique acid–base center. Moreover, the hydropho-
bic interior of enzyme active sites that undergo domain closure
can greatly perturb the pKa values of amino acid side chains rela-
tive to their typical pKa values in aqueous solution. At any rate,
site-directed mutagenesis of Arg64, Arg94, and Arg138 residues
of hUP1 and crystal structure determination of these mutants will
have to be carried out to ascertain the role, if any, of these residues
in Pi binding.
The pH dependence of kcat/KUrd (Fig. 7C) indicates that proton-
ation of group with pKa of 6.5 ( 0.6) and deprotonation of another
group with pKa of 8.4 ( 0.9) abolish uridine binding. The side
chains of His8 and Glu198 have been shown to be involved in E. coli
UP ribose binding site [18], corresponding to His36 and Glu250 in
hUP1. The side chain of His36 is a more likely candidate for the
group with pKa value of 6.5 that has to be deprotonated to interact
with the 50-OH group of the ribose moiety of Urd in hUP1. There
are also H-bonds formed between the 20-OH group of ribose and
the main-chain nitrogen of Met197 (Met249 in hUP1) and the side
chain of Arg91 (Arg138 in hUP1) [18]. As Arg138 has been shown
to be involved in Pi binding in hUP1 [7], it is not likely that
Fig. 5. Dissociation constant for hUP1 with Pi (A) and with R1P (B) binary complex
formation monitoring changes in intrinsic protein fluorescence. Insets represent the
fit of double-reciprocal plots of the fluorescence data to an exponential growth
equation.
5-FU and R1P or FUrd and Pi, it has been proposed that there ap-
pears to be a cooperative pattern of substrate binding [18]. Our re-
sults demonstrate that there is indeed cooperativity that is brought
about by Pi or R1P binding to hUP1.
The initial velocity, product inhibition, and equilibrium binding
results are consistent with a steady-state ordered bi bi kinetic
mechanism, in which Pi binds first to the free enzyme, followed
by the binding of Urd to form the catalytically competent ternary
complex, and uracil is the first product to dissociate from the com-
plex, followed by the release of R1P (Fig. 6).
pH-rate profiles
pH dependence of the kinetic parameters was evaluated to
probe acid–base catalysis in hUP1 mode of action. The bell-shaped
pH-rate profiles were fitted to the following equation: log y =
log[C/(1 + H/Ka + Kb/H)], yielding Ka and Kb, respectively, the appar-
ent acid and base dissociation constants for ionizing groups. In this
equation, y represents the apparent kinetic parameter (kcat or kcat
/
KM), C is the pH-independent plateau value of y, and H is the proton
concentration. The bell-shaped pH-rate profiles showed values of 1
for the acidic limb and ꢀ1 for the basic limb, indicating participa-
Fig. 6. Proposed kinetic mechanism for hUP1.

D. Renck et al. / Archives of Biochemistry and Biophysics 497 (2010) 35–42
41
(Arg219 in hUP1) interacts with the O4 of the carbonyl group of
uracil, and it has been proposed to play a role in substrate selectiv-
ity via an electrostatic effect [18]. It is thus tempting to assign to
the guanidinium side chain of Arg219 residue the pKa value of
8.4 that has to be protonated for Urd binding to occur. Notwith-
standing, site-directed mutagenesis will have to be carried out to
assign any role in substrate binding and catalysis to a particular
amino acid residue in hUP1.
Summary
Here we describe an efficient method to obtain homogeneous
recombinant hUP1. We also present initial velocity, product inhibi-
tion, and equilibrium binding data which show that hUP1 catalyzes
the phosphorolysis of Urd by a steady-state ordered bi bi kinetic
mechanism, in which Pi binds first to free enzyme, followed by
the binding of Urd to form the catalytically competent ternary
complex, and uracil is the first product to dissociate from the com-
plex, followed by R1P release. Amino acid residues involved in
either catalysis or substrate binding were proposed based on pH-
rate profiles. A comparison between the crystal structure of free
hUP1 and BAU-hUP1 binary complex showed that there is a large
inter-domain motion between monomers of dimeric hUP1 [7].
These findings prompted the authors to propose that the ‘‘open”
conformation of hUP1 provides an opportunity to develop a novel
class of allosteric inhibitors of this enzyme that lock the protein in
a functionally disabled form [7]. However, there was no experi-
mental evidence showing that hUP1 exhibits allostery. Thermody-
namic dissociation constants were assessed by the enhancement of
intrinsic protein fluorescence upon Pi or R1P binding to hUP1, and
the results demonstrated that these substrates exhibit cooperative
binding to the enzyme. These results lend support to the proposal
of designing allosteric inhibitors of hUP1 enzyme activity. It has
been proposed that the conservation of key residues and interac-
tions with substrate in the phosphate and ribose binding pockets
would indicate that ribooxocarbenium ion formation during catal-
ysis of UP may be similar to that proposed for E. coli purine nucle-
oside phosphorylase (PNP) [18]. Human PNP catalyzes the
phosphorolysis of N-ribosidic bonds of 6-oxy-purine nucleosides
and deoxynucleosides to the corresponding purine base and a-D-ri-
bose 1-phosphate. Transition-state analogues that have picomolar
inhibition constants have been developed for human PNP based on
the transition-state structure for calf spleen PNP [45]. For instance,
Immucillin-H possesses features of the transition state which in-
clude an elevated pKa at the N7 position of the 9-deazahypoxan-
thine ring,
a positive charge in the protonated iminoribitol
moiety to mimic the ribooxocarbenium ion, and an enzymatically
stable carbon–carbon ribosidic bond [45]. Human PNP has a later
transition state and DADMe-Immucillin-H has been synthesized
to be a mimicry of the proposed transition state. On the other hand,
bovine PNP has an earlier transition state and Immucillin-H has
been synthesized to be a mimicry of this transition state. It has
been shown that DADMe-Immucillin-H binds more tightly to hu-
man PNP than Immucillin-H, thereby showing that even though
bovine and human PNPs share 87% sequence identity and have to-
tally conserved active site residues, inhibitors with differential
specificity can be designed [46]. The crystal structure of human
PNP in complex with Immucillin-H showing the amino acid resi-
dues that interact with this transition-state analogue has been re-
ported [47]. Accordingly, the transition-state analogues of human
PNP could serve as blueprints for the design of inhibitors of hUP1
enzyme activity. As hUP1 is a molecular target for the design of
specific inhibitors intended to boost endogenous uridine levels
for the purpose of rescuing normal tissues from the toxicity of flu-
oropyrimidine nucleoside chemotherapeutic agents, the data here
Fig. 7. pH dependence of hUP1 kinetic parameters. Plots: (A) log kcat
log kcat=KP , and (C) log kcat/KUrd
, (B)
.
i
Arg138 represents the group with pKa of 8.4 whose deprotonation
abolishes Urd binding. The crystal structure of E. coli UP has also
shown that Gln166 and Arg168 are key residues in the uracil bind-
ing pocket and together with a tightly bound water molecule are
seen to be involved in the substrate specificity of UP [18]. These
residues correspond to Gln217 and Arg219 in human UP1 [7]. It
is unlikely that this role is played by Gln217 because its amine side
chain does not ionize. On the other hand, the d-guanidinium group
of the side chain of the conserved Arg168 amino acid in E. coli UP

42
D. Renck et al. / Archives of Biochemistry and Biophysics 497 (2010) 35–42
[21] J.G. Robertson, Biochemistry 44 (2005) 5561–5571.
[22] J.G. Robertson, Curr. Opin. Struct. Biol. 17 (2007) 674–679.
[23] U.K. Laemmli, Nature 227 (1970) 680–685.
[24] M.M. Bradford, Anal. Biochem. 72 (1976) 248–254.
[25] B.M. de Souza, M.S. Palma, Biochim. Biophys. Acta 1778 (2008) 2797–2805.
[26] H. Chassaigne, R. Lobinski, Analyst 123 (1998) 2125–2130.
[27] G. Magni, Methods Enzymol. 51 (1978) 290–296.
[28] P.F. Cook, W.W. Cleland, Enzyme Kinetics and Mechanisms, Garland Science,
London, New York, 2007.
presented provide pivotal data for the design of function-based
inhibitors. Understanding the mode of action of hUP1 will inform
us on how to better design inhibitors targeting hUP1 with potential
therapeutic application in cancer chemotherapy.
References
[1] G. Pizzorno, D. Cao, J.J. Leffert, R.L. Russel, D. Zhang, R.E. Handschumacher,
Biochim. Biophys. Acta 1587 (2002) 133–144.
[29] K.C. Kelley, K.J. Huestis, D.A. Austen, C.T. Sanderson, M.A. Donohue, S.K. Stickel,
E.S. Kawasaki, M.S. Osburne, Gene 156 (1995) 33–36.
[2] M. Johansson, Biochem. Biophys. Res. Commun. 307 (2003) 41–46.
[3] S.I. Watanabe, T. Uchida, Biochem. Biophys. Res. Commun. 216 (1995) 265–
272.
[30] C. Rizzi, J. Frazzon, F. Ely, P.G. Weber, I.O. Fonseca, M. Gallas, J.S. Oliveira, M.A.
Mendes, B.M. Souza, M.S. Palma, D.S. Santos, L.A. Basso, Protein Expr. Purif. 40
(2005) 23–30.
[4] M.J. Pugmire, S.E. Ealick, Biochem. J. 361 (2002) 1–25.
[5] D. Cao, J.J. Leffert, J. McCabe, B. Kim, G. Pizzorno, J. Biol. Chem. 280 (2005)
21169–21175.
[6] D. Cao, R.L. Russell, D. Zhang, J.J. Leffert, G. Pizzorno, Cancer Res. 62 (2002)
2313–2317.
[31] R.G. Silva, L.P. Carvalho, J.S. Oliveira, C.A. Pinto, M.A. Mendes, M.S. Palma, L.A.
Basso, D.S. Santos, Protein Exp. Purif. 27 (2003) 158–164.
[32] T.H. Grossman, E.S. Kawasaki, S.R. Punreddy, M.S. Osburne, Gene 209 (1998)
95–103.
[33] F.W. Studier, Protein Expr. Purif. 41 (2005) 207–234.
[34] C.H. Schein, Biotechnology 7 (1989) 1141–1149.
[35] P.H. Hirel, M.J. Schmitter, P. Dessen, G. Fayat, S. Blanquet, Proc. Natl. Acad. Sci.
86 (1989) 8247–8251.
[36] I.H. Segel, Enzyme Kinetics, Behavior and Analysis of Rapid Equilibrium
and Steady-state Enzyme Systems, John Wiley and Sons, Inc., New York,
1975.
[37] A. Kraut, E.W. Yamada, J. Biol. Chem. 246 (1971) 2021–2030.
[38] T.A. Krenitsky, Biochim. Biophys. Acta 429 (1976) 352–358.
[39] A. Vita, C.Y. Huang, G. Magni, Arch. Biochem. Biophys. 226 (1983) 687–692.
[40] Y. Avraham, N. Grossovicz, J. Yashphe, Biochem. Biophys. Acta 1040 (1990)
287–293.
[7] T.P. Roosild, S. Castronovo, M. Fabbiani, G. Pizzorno, BMC Struct. Biol. 16 (2009)
9–14.
[8] D.B. Longley, D.P. Harkin, P.G. Johnston, Nat. Rev. Cancer 3 (2003) 330–338.
[9] E.Y. Morgunova, A.M. Mikhailov, A.N. Popov, E.V. Blagova, E.A. Smirnova, B.K.
Vainshtein, C. Mao, S.R. Armstrong, S.E. Ealick, A.A. Komissarov, E.V. Linkova,
A.A. Burlakova, A.S. Mironov, V.G. Debabov, FEBS Lett. 367 (1995) 183–187.
[10] M. Liu, D. Cao, R. Russell, R.E. Handschumacher, G. Pizzorno, Cancer Res. 58
(1998) 5418–5424.
[11] H. Miyashita, Y. Takebayashi, J.F. Eliason, F. Fujimori, Y. Nitta, A. Sato, H.
Morikawa, A. Ohashi, K. Motegi, M. Fukumoto, S. Mori, T. Uchida, Cancer 94
(2002) 2959–2966.
[12] T.A. Krenitsky, M. Barclay, J.A. Jacquez, J. Biol. Chem. 239 (1964) 805–812.
[13] D. Cao, M.A. Nimmakayalu, F. Wang, D. Zhang, R.E. Handschumacher, P. Bray-
Ward, G. Pizzorno, Cancer Res. 59 (1999) 4997–5001.
[14] Y. Im, H.K. Shin, H. Kim, S. Jeong, S. Kim, Y. Kim, D.H. Lee, S. Jeon, H. Lee, J. Choi,
Mol. Cells 28 (2009) 119–124.
[41] T.A. Krenitsky, J. Biol. Chem. 243 (1968) 2871–2875.
[42] A.V. Hill, Biochem. J. 7 (1913) 471–480.
[43] N.M. Goodey, S.J. Benkovic, Nat. Chem. Biol. 4 (2008) 474–482.
[44] R.A. Copeland, Enzymes: A Practical Introduction to Structure, Mechanism, and
Data Analysis, Wiley-VCH, New York, 2000.
[15] B. Deneen, H. Hamidi, C.T. Denny, Cancer Res. 63 (2003) 4268–4274.
[16] A. Arvand, C.T. Denny, Oncogene 20 (2001) 5747–5754.
[17] D. Zhang, D. Cao, R. Russell, G. Pizzorno, Cancer Res. 61 (2001) 6899–6905.
[18] T.T. Caradoc-Davies, S.M. Cutfield, I.L. Lamont, J.F. Cutfield, J. Mol. Biol. 337
(2004) 337–354.
[45] V.L. Schramm, Arch. Biochem. Biophys. 433 (2005) 13–26.
[46] E.A. Taylor Ringia, P.C. Tyler, G.B. Evans, R.H. Furneaux, A.S. Murkin, V.L.
Schramm, J. Am. Chem. Soc. 128 (2006) 7126–7127.
[47] W.F. Azevedo Jr., F. Canduri, D.M. Santos, J.H. Pereira, M.V. Dias, R.G. Silva, M.A.
Mendes, L.A. Basso, M.S. Palma, D.S. Santos, Biochem. Biophys. Res. Commun.
309 (2003) 917–922.
[19] R. Bose, E.W. Yamada, Biochemistry 13 (1974) 2051–2056.
[20] G.P. Connolly, J.A. Duley, Trends Pharmacol. Sci. 20 (1999) 218–225.