Reaction of human UMP-CMP kinase with naturaland analog substrates Claudia Pasti1,*,†, Sarah Gallois-Montbrun1,†, He´le`ne Munier-Lehmann2, Michel Veron1, Anne-Marie Gilles2 and Dominiqu
Trang 1Reaction of human UMP-CMP kinase with natural
and analog substrates
Claudia Pasti1,*,†, Sarah Gallois-Montbrun1,†, He´le`ne Munier-Lehmann2, Michel Veron1, Anne-Marie Gilles2 and Dominique Deville-Bonne1
1
Unite´ de Re´gulation Enzymatique des Activite´s Cellulaires and2Laboratoire de Chimie Structurale des Macromole´cules,
CNRS URA 2185, Institut Pasteur, Paris, France
UMP-CMP kinase catalyses an important step in the
phosphorylation of UTP, CTP and dCTP It is also involved
in the necessary phosphorylation by cellular kinases of
nucleoside analogs used in antiviral therapies The reactivity
of human UMP-CMP kinase towards natural substrates
and nucleotide analogs was reexamined The expression of
the recombinant enzyme and conditions for stability of the
enzyme were improved Substrate inhibition was observed
for UMP and CMP at concentrations higher than 0.2 mM,
but not for dCMP The antiviral analog L-3TCMP was
found to be an efficient substrate phosphorylated into
L-3TCDP by human UMP-CMP kinase However, in the reverse reaction, the enzyme did not catalyse the addition of the third phosphate to L-3TCDP, which was rather an inhibitor By molecular modelling,L-3TCMP was built in the active site of the enzyme from Dictyostelium Human UMP-CMP kinase has a relaxed enantiospecificity for the nucleoside monophosphate acceptor site, but it is restricted
toD-nucleotides at the donor site
Keywords: UMP-CMP kinase; antiviral analog; 3TC; AraC; phosphorylation
Nucleoside analogs constitute a familly of important
antiviral and anticancer drugs Analogs of thymidine like
AZT (2¢3¢-deoxy-3¢azido thymidine), d4T
(2¢3¢-dideoxy-2¢3¢-didehydro-thymidine) have been used to treat HIV infection
for a number of years as well as analogs with an
L-conformation in the sugar likeL-3TC All of these analogs
are delivered as nucleosides to the patients and need to be
phosphorylated within cells in order to be active on their
viral or cellular target UMP-CMP kinase catalyses the
reversible transfer of phosphate between UMP (or CMP)
and ATP in the presence of magnesium ions according to
the followingreaction:
UMP þ ATP-Mg2þ ! UDP þ ADP-Mg 2þ The enzyme is involved in both the de novo and the salvage pathway of nucleosides It catalyses a compulsory step for the synthesis of UTP, CTP and dCTP, that are incorporated into nucleic acids UMP-CMP kinase is therefore also involved in the activation of nucleoside analogs and its kinetic properties are important for the efficacy and the selectivity of these drugs The UMP-CMP kinase of several eukaryotic organisms has been studied including yeast [1], the slime mold Dictyostelium discoideum [2,3] and, recently,
in man [4] The UMP-CMP kinase mRNA was ubiqui-tously present in human tissues with highest levels in pancreas, muscle and liver [4] The enzyme belongs to the large family of eukaryotic nucleoside and NMP kinases for which AMP kinase is a prototype [5,6] In contrast, in prokaryotes and, in particular in E coli, UMP kinase and CMP kinase are different proteins sharingno sequence similarity [7,8] The 3D structure of UMP-CMP kinase from the Saccharomyces cerevisiae [9] and from D discoid-eum [3] have been solved by X-ray crystallography and compared to the structure of yeast S cerevisiae adenylate kinase [10] All NMP kinases are globular proteins with a
core domain made of a five-stranded parallel b-sheet surrounded by eight helices The protein is made of two subdomains, each containinga nucleotide bindingsite The site for the nucleotide donor of phosphate (in general ATP) presents a typical P-loop and a lid domain that closes down over the active site duringthe catalytic cycle The protein, which is flexible as several structural changes occur upon ligand binding, moves from an open to a closed conformation duringthe catalytic cycle [10]
UMP-CMP kinase is involved in the phosphorylation
of several anticancer and antiviral drugs that are given
to patients sufferingfrom AIDS and B hepatitis
Correspondence to D Deville-Bonne, Institut Pasteur, Unite´ de
Re´gulation Enzymatique des Activite´s Cellulaires, 25, rue du Dr Roux,
75724 Paris Cedex 15, France.
Fax: + 33 1 45 68 83 99, Tel.: + 33 1 40 61 35 35,
E-mail: ddeville@pasteur.fr
Abbreviations: AraC, cytosine D -arabinofuranoside; AZT,
2¢,3¢-deoxy-3¢-azido thymidine; GST, glutathione S-transferase; dCK,
deoxycyti-dine kinase; L -dC, b- L -2¢-deoxcytidine; d4T,
2¢3¢-dideoxy-2¢3¢-didehydro-thymidine; L -dT, b- L -2¢-deoxythymidine; L -3TC,
b- L -2¢,3¢-dideoxy-3¢-thiacytidine; L -3TCMP, b- L
-2¢,3¢-dideoxy-3¢-thi-acytidine monophosphate; L -3TCDP, b- L
-2¢,3¢-dideoxy-3¢-thiacyti-dine diphosphate; L -3TCTP, b- L -2¢,3¢-dideoxy-3¢-thiacytidine
triphosphate; NMP, nucleoside monophosphate; NDP, nucleoside
diphosphate; NTP, nucleoside triphosphate.
Enzyme: Human UMP-CMP kinase (EC 2.7.4.14).
*Present address: Universita deg li Studi di Ferrara, Dip Biochimica e
Biologia Molecolare,Via Borsari, 46, 44100 Ferrara, Italy.
Note: Both authors contributed equally to this work.
(Received 18 December 2002, revised 19 February 2003,
accepted 25 February 2003)
Trang 2Arabinofuranosylcytocine (AraC) has a hydroxyl group in
the trans configuration at the 2¢-position of the sugar b-L
-2¢-deoxynucleosides were shown to inhibit specifically hepatitis
B replication, alongwith b-L-thymidine (L-dT) and b-L
-deoxycytidine (L-dC) which are the most potent inhibitors
[11] b-L-NTP and b-D-NTP are enantiomers resultingfrom
the asymmetry of the C4¢ and C1¢ of the ribose.L-cytidine
analogs used in therapies are substrates of deoxycytosine
kinase This enyzme, which adds the first phosphate to
cytidine derivatives with variable sugar structures, has a low
enantioselectivity although it generally favors theD
-enantio-mer [12] Human UMP/CMP kinase was recently shown
to phosphorylate monophosphate derivatives of
deoxycyti-dine analogs in either theL- orD- conformers [13] The third
phosphate addition to L-cytidine analogs is catalysed by
phosphoglycerate kinase [14] but not by NDP kinase, which
reacts only withD-nucleotide analogs [15] Another NMP
kinase, AMP kinase also called myokinase, was reported to
complement NDP kinase deficiency in E coli upon gene
disruption [16] AMP kinase catalyses the reversible
ATP-dependent synthesis of ADP from AMP Therefore the
reverse reaction forms ATP and AMP from two ADP
molecules We ask the question whether UMP-CMP kinase
also allows the addition of the third phosphate to analog
diphosphate substrates in the reverse reaction We analyze
here the specificity of human UMP-CMP kinase for
different substrates including L-3TCMP in the forward
and backward reactions
Materials and methods
Chemicals
All nucleotides were purchased from Sigma chemicals
(St Louis, MO, USA).L-3TC was extracted from Epivir
tablets (GlaxoWellcome) with methanol After purification
by silica gel column chromatography (dichloromethane/
methanol, 95 : 5, v/v), the nucleoside was phosphorylated
into the 5¢-monophosphate by overnight incubation at
20C with GTP in the presence of
deoxyadenosine-deoxycytidine kinase from Bacillus subtilis (a kind gift
of J Neuhard, University of Copenhagen, Denmark)
L-3TCMP was purified by successive chromatography on a
G-10 column (240 mL total volume) in water and by reverse
phase on RP-18 silica gel (50 mL) The purity ofL-3TCMP
was characterized by nuclear magnetic resonance (1H,31P)
spectroscopy and mass spectrometry
Cloning of the gene coding for UMP-CMP kinase,
expression and purification of His-tagged
UMP-CMP kinase
The 588-bp fragment corresponding to the gene coding for
the human UMP-CMP kinase was amplified by PCR [17]
usingcDNA from SK melanoma cells as the matrix The
two synthetic oligonucleotides used for amplification were:
5¢-GGAATTCCATATGAAGCCGCTGGTCGTGTTC
GTCCTCGGCGGCCCCGGCGCCGGCAAGGGGA
CC-3¢ and 5¢-CCGCTCGAGTTAGCCTTCCTTGTCA
AAAATCTG-3¢ Duringamplification, NdeI and XhoI
restriction sites (in bold letters in the oligonucleotide
sequences) were created at both ends of the amplified
fragment After digestion by NdeI and XhoI, the amplified gene was inserted into the pET28a plasmid (Novagen, Inc.) digested with the same enzymes Two clones containingthe gene codingfor the human UMP-CMP kinase and overexpressingthe human UMP-UMP-CMP kinase with a His-tagat the N-terminal part were characterized One of them was kept for further studies and the correspondingplasmid named pHL60-5 The DNA insert was sequenced [18] in order to verify the absence of any mutational events in the course of amplification
BL21 (DE3)/pDIA17 E coli strain [19] transformed with pHL60-5 plasmid was grown in 2YT medium [17] contain-ing30 lgÆmL)1 chloramphenicol and 70 lgÆmL)1 kana-mycine until D600¼ 1 After induction with 1 mMisopropyl thio-b-D-galactoside and growth for 3 h at 30C, cells were harvested, resuspended in 40 mL lysis phosphate buffer
50 mMpH 8 containing300 mMNaCl, 10 mMimidazole, protease inhibitors (Complete EDTA-free, Roche) and
1 mMdithiothreitol and kept at)80 C Cells were broken
in a French Press at 100 Mpa and centrifuged for 30 min at
12 000 g at 4C The supernatant was added onto a 20-mL Ni-nitrilotriacetic acid column (Qiagen, Germany) preequili-brated with lysis buffer The column was washed with lysis buffer (100 mL) The protein was then eluted by a linear imidazole gradient (10–250 mM) pH 8.0 Fractions contain-ingthe enzymatic activity were pooled and dialyzed against Tris/HCl 50 mM pH 7.5 containing20 mM NaCl, 1 mM dithiothreitol and 50% glycerol and then kept at)20 C The protein was > 99% pure as judged by SDS/PAGE on 15% acrylamide gels (Biorad)
The homogeneity of the enzyme preparation was meas-ured by dynamic light scatteringexperiments usinga Dynapro-800 instrument (Proteinsolutions) The protein was > 99.6% monomer with a hydrodynamic radius of 2.2 nm and a molecular mass estimated as 22 000 The Stokes radius was measured by gel filtration on a Superdex 75H 10/30 (Amersham Biosciences) in Tris/HCl 50 mM,
pH 7.5 containing200 mM KCl and 1 mM dithiothreitol, usingbovine albumin, ovalbumin and chymotrypsinogen as markers The elution volume of UMP-CMP kinase indica-ted a Stokes radius of 2.8 nm, in agreement with a monomer species
Expression and purification of human UMP-CMP kinase
as a fusion protein with GST BL21 gold (DE3) E coli cells (Stratagene) were transformed with the plasmid umpk-pGEX5X, a kind gift from A Kar-lsson (Karolinska Institute, Stockholm, Sweden), codingfor
a fusion protein with the GST at the N-terminus of UMP-CMP kinase [4] Cells were grown in 2YT medium [17] containingampicillin 100 lgÆmL)1until D600¼ 0.4 After induction with 1 mM isopropyl thio-b-D-galactoside and growth for 5 h at 22C, cells were harvested, washed once
in 100 mM Tris/HCl pH 8 containing10 mM MgCl2 and
1 mMdithiothreitol, resuspended in 50 mMTris/HCl pH 8 containing1 mMEDTA, 5 mMMgCl2, 5 mMKCl, 1 mM dithiothreitol and 0.5 mMphenylmethanesulfonyl fluoride and kept at )80 C The cells were broken as indicated above, centrifuged for 30 min at 12 000 g and the super-natant was incubated with glutathione-agarose for 1 h at
Trang 34C accordingto the Amersham Biosciences procedure.
The gel was poured into a column and washed with
phosphate buffer 100 mMpH 7.4 containing100 mMNaCl,
1 mM EDTA and 1 mM phenylmethanesulfonyl fluoride
The fusion protein was eluted by phosphate buffer 100 mM
pH 7.4 containing100 mMNaCl, 5 mMMgCl2and 10 mM
glutathione The fractions containing UMP-CMP kinase
activity were pooled; glycerol was added to a 20% final
concentration and kept at )20 C The protein was
found 95% pure by SDS/PAGE on 15% acrylamide gels
(Biorad) No loss in activity was observed after 2 months
The typical yield was 10 mgpure fusion protein per litre of
culture
Expression and purification ofDictyostelium UMP-CMP
kinase
Plasmid pIMS5, a kind gift from L Wiesmu¨ller (University
of Hamburg, Germany), was expressed in XL1Blue E coli
strain in Luria Broth After growth at 37C to D600¼ 0.5,
isopropyl b-D-thiogalactoside (0.5 mM) induction was
car-ried out for 4 h at 22C After centrifugation, cells were
resuspended in 50 mM Tris/HCl buffer pH 7.4 containing
1 mMEDTA and 2 mMdithiothreitol (buffer A) The cells
were sonicated (3· 2 min) and centrifuged (17 000 g,
30 min) The supernatant was applied onto a
Blue-Seph-arose column (Amersham Biosciences) (100 mgprotein for
6 mL of gel), washed with 50 mL of buffer A and 10 mL of
buffer A containing0.3M NaCl, before elution of the
protein with buffer A containing1M NaCl Further
purification by gel filtration chromatography (Ultrogel
AcA 54, Sigma) in buffer A without dithiothreitol resulted
in pure enzyme It was stored at)20 C in 20% glycerol
Enzymatic activity of UMP-CMP kinase
The forward reaction of UMP-CMP kinase was followed in
a spectrophotometer by measuringADP formation as
described [20] in the presence of dithiothreitol [4,13,21] The
assay contained 50 mM Tris/HCl pH 7.4, 50 mM KCl,
5 mMMgCl2, 1 mMATP, 0.2 mMNADH, 10 mM
dithio-threitol, 1 mM phosphoenolpyruvate, 1 mM ATP and the
auxiliary enzymes: pyruvate kinase (4 U), lactate
dehydro-genase (4 U) and NDP kinase from Dictyostelium (4 U) In
the case of L-3TCMP as substrate, no NDP kinase was
added, asL-3TCDP is not a substrate for this enzyme [15]
The reaction was started at 37C by addition of 1 mM
dCMP or another phosphate acceptor (1 mM) and enzyme
(final concentration 8.4 lgÆmL)1¼ 0.17 lM) In order to
avoid limitation by the coupled system, the rates were below
0.2 units at A340per min The rate was calculated assuming
that two ADP are generated during the reaction (due to the
presence of NDP kinase), except whenL-3TCMP was the
phosphoacceptor where only one ADP was produced
Curve-fit was performed using KALEIDAGRAPH (Abelbeck
Software) for a hyperbolic progress equation unless
indica-ted Alternatively the enzyme-coupled assay was automated
in 96-wells microplates in 250 lL final volume with four
times less enzyme and the reactions were monitored during
10 min on a microplate reader (BIO-TEK Elx808) The
reaction rates were calculated usingtheKC4software
(BIO-TEK Instruments, USA)
The reverse reaction catalysed by the human UMP-CMP kinase was assayed with [3H]CDP (Amersham Biosciences) and CDP, dCDP orL-3TCDP as substrates The formation of dCTP (orL-3TCTP) catalysed by UMP-CMP kinase was studied under steady state conditions The amount of [3H]CMP and [3H]CTP formed in the presence of 1 mM [5-3H]CDP (0.2 CiÆmmol)1) and 0.25–
2 mM of dCDP orL-3TCDP was monitored for 2, 4 and
6 min The assays were started by addingthe enzyme (final concentration 8.4 lgÆmL)1¼ 0.17 lM) to a reaction mixture (15 lL) containing50 mM Tris/HCl pH 7.5,
5 mM MgCl2 and the substrates at 37C The reaction was stopped at 2, 4 and 6 min by adding3 lL aliquots
of the reaction mixture to 2 lL stop solution consisting
of 1 mMformic acid and 10 mMeach of CDP and CTP Radioactive CMP, CDP and CTP were separated on TLC aluminum sheets (Silica gel 60 F254, Merck), which were developed with isopropanol/NH3 30%/H2O,
3 : 1 : 1, v/v/v After drying, the plates were counted on
a BETA-Imager 2000 counter (Biospace, Paris, France) [21] and analyzed As [3H]CMP was made in parallel in the reaction from two [3H]CDP molecules, the corres-pondingbackground was subtracted from the results obtained in the presence of dCDP orL-3TCDP
Results
Analysis of the forward reaction catalysed
by UMP-CMP kinase with natural substrates and nucleoside analogs
The purified recombinant human UMP-CMP kinase expressed as a His-tagfusion was analyzed by dynamic light scattering and gel filtration on Superdex 75, yielding an hydrodynamic radius of 2.2 nm and 2.8 nm, respectively (results not shown) This corresponded to a monomeric state of the protein Figure 1A and Table 1 show the activity of human UMP-CMP kinase as a function of natural substrates, and the correspondingkinetic param-eters The reaction rates were found to increase as a function
of UMP and CMP as expected for Michaelis curves, but did not reach a plateau (the maximum rate V) due to substrate inhibition at concentrations above 0.2 mM The correpond-inginhibition constants Ki were 0.5 mM for CMP and 1.5 mM for UMP, with Km values of 22 lM and 44 lM, respectively In contrast, no excess substrate inhibition was found with dCMP (Km¼ 0.9 mM), indicatingthat the presence of the 2¢OH of the ribose is presumably involved in the inhibition mechanism Both UMP and CMP were found
to be better substrates than dCMP with smaller Kmand higher V, resultingin considerably improved catalytic efficiencies (kcat/Km) These data were found for concentra-tions of 5 mMMg2+ions and above
The substrate specificity of the His-tagged enzyme was investigated by assaying a series of nucleoside monophos-phates: AMP was found to be a slow substrate with Km higher than 5 mM and a catalytic efficiency about
1000M )1Æs)1while dTMP and GMP were not substrates (kcat/Km< 100M )1Æs)1) (not shown) The activity of human UMP-CMP kinase was then analyzed usingnucleo-side analogs as substrates As shown in Fig 1B, (L)-3TCMP and AraCMP were substrates with K in the 0.2 mMrange
Trang 4No inhibition by excess of substrate was detected The
turnover number for AraCMP was similar to that of UMP
(around about 150 s)1) and it was significantly lower for
L-3TCMP (36 s)1) (Table 1) Accordingto catalytic
effi-ciencies, NMPs fall in two groups with catalytic efficiencies
in the rang e of 107M )1Æs)1for UMP and CMP, and of
105M )1Æs)1for AraCMP,L-3TCMP and dCMP (Table 1) Careful characterization of the reaction catalysed by human UMP-CMP kinase indicated that the type of fusion protein made for purification purposes could interfere with the kinetic properties of the enzyme Thus, when expressed
as a GST fusion, the kinetic parameters of human UMP-CMP kinase were different from those of the His-tagged fusion protein As shown in Table 2, the catalytic efficiencies calculated from Kmand kcatfor both natural substrates or analogs resulted in an average fivefold decrease in catalytic efficiencies as compared to the His-tagged enzyme shown in Table 1 As NMP kinases are very flexible proteins [10], this decrease in activity with the GST-fusion enzyme is likely to correspond to a decrease in the protein dynamics due to the presence of the bulky additional GST domain
We also compared the kinetic properties of the human UMP-CMP kinase with those of the enzyme from Dicty-osteliumwhose tertiary structure was determined by X-ray crystallography [3] (Table 3) Although both proteins show 50% sequence identity, several differences in the kinetic properties were observed (Table 3) For natural substrates (UMP and CMP), no inhibition by substrate excess is found While the turnover is increased for the Dictyostelium enzyme by two fold, a decrease in catalytic efficiencies from
107M )1Æs)1for human UMP-CMP kinase to 106M )1Æs)1is observed for the Dictyostelium enzyme Table 3 shows that DictyosteliumUMP-CMP kinase also acceptsL-3TCMP as substrate, although it reacts with the analog about 500 times less than the human enzyme
The Dictyostelium enzyme seemed a more appropriate structural model for the human enzyme [3,22] than the protein from yeast, which differs significantly in specificity since it also reacts with adenosine [23,24] The structure of DictyosteliumUMP-CMP kinase has been determined as a complex with ATP, CMP as well as with AlF3 (PDB accession no 3UKD), providinga model for the active state, as aluminum fluoride resembles a transition state of a phosphoryl transfer reaction [22] Figure 2 shows a model in which CMP was replaced byL-3TCMP in the acceptor site
of Dictyostelium UMP-CMP kinase Startingfrom the superimposition of the a-phosphates, the oxathiolan ringof
L-3TCMP could fit over the CMP ribose This results in a translation of the cytosine ringof about 1.2 A˚ This shift seems to be easily tolerated by the bindingsite, due to the absence of direct interactions between the base moiety and the side chains of the enzyme [3] In conclusion,L-3TCMP can be modeled into the CMP bindingsite of UPM-CMP
Fig 1 Measure of the steady-state kinetics parameters of human
UMP-CMPkinase (A) Kinetic for natural substrates UMP, CMP, dCMP.
(B) Kinetic for substrate analogs D-AraCMP and L -3TCMP V is
expressed in unit (lmol productÆmin)1)Æmg)1 of the fusion protein
UMP-CMP kinase with GST (PM ¼ 51 000) The reaction rate v with
UMP and CMP as a substrate [S] were best fitted with Eqn (1).
v ¼ V S½
K m þ S ½ þ½ sKi2
ð1Þ
Table 1 Kinetic parameters of human His-tagged UMP-CMP kinase in the forward reaction Activity measurements were carried out usinga spectrophotometric coupled assay under initial rates conditions in the presence of 1 m M ATP and 5 m M Mg2+as described in Material and methods section (conditions of Fig 1) Values are means of two to four independent measurements Even when the Michaelis curve reached a plateau, catalytic efficiencies have been calculated from the linear fit of the first values (initial rate as a function of substrate concentration).
No inhibition
No inhibition
Trang 5kinase, confirmingthe absence of stereospecificity observed
at the acceptor site level
The UMP-CMP kinase reverse reaction
We have examined the possibility that UMP-CMP kinase
could also catalyse the addition of the third phosphate in the
reverse reaction (see scheme above) Clearly this would
depend on the stereospecificity of the donor site The terms
acceptor and donor are used here accordingto the forward
reaction, with the donor site bindinga nucleotide
triphos-phate (ATP but also other NTPs) and the acceptor site
bindinga nucleotide monophosphate (preferentially CMP
and UMP) In the reverse reaction, both donor and
acceptor sites bind a nucleotide diphosphate We have
studied this reaction using[3H]CDP as a substrate Product
formation was measured with a BETA Imager after
separation on TLC plates as described under Materials
and methods Under our experimental conditions, when [3H]CDP was used alone as substrate, both [3H]CMP and [3H]CTP were produced in similar amounts (Fig 3A, lane 2–4) Because no radioactiveL-3TCDP was available, we
Table 2 Kinetic parameters of human GST fusion UMP-CMP kinase in the forward reaction Activity measurements were carried out under the conditions described in Table 1.
0.5 ± 0.2
No inhibition
Table 3 Kinetic parameters of Dictyostelium UMP-CMP kinase in the forward reaction Activity measurements and analysis were carried out under the conditions described in Table 1 except for the determination of catalytic efficiencies which obtained from the ratio of the kinetic parameters k cat and K m
ðk cat =K m Þhuman
0.30
Fig 2 Illustrations of L -3TCMPand CMPsuperimposed into the
acceptor site in Dictyostelium UMP-CMP kinase The dockingwas
performed on a Silicon Graphics workstation using O software [33].
CMP is represented accordingto the conformation in the complex with
the kinase as found in 3UKD.PDB b- L -3TCMP was positioned in the
active site by choosingthe same orientation for the cytidine base
moiety as in the complex with CMP Carbon atoms of CMP and
L -3TCMP are in black and g rey, respectively, with sulfur in yellow,
phosphorus in magenta and oxygens in blue (for the base) and red (for
the sugar and the phosphate) Left, overview on the base moiety; right,
overview on the sugar moiety.
Fig 3 Kinetic of backward reaction of His-tagged human UMP-CMP kinase (A) Time-course of products formation after separation on a TLC silica plate visualized on the BETA Imager The substrates are [3H]CDP plus CDP (lanes 2–4), or plus dCDP (lanes 5–7) or plus
L -3TCDP (lanes 8–10) (B) Initial rates of [ 3 H]CMP formation upon addition of dCDP or -3TCDP.
Trang 6developed a competitive assay in order to analyze the ability
of the enzyme to use nucleotide analogs in the reverse
reaction Indeed when an unlabelled NDP was added to
[3H]CDP, the reaction was expected to produce [3H]CMP
and NTP and/or [3H]CTP and NMP in equal amounts The
relative affinities of the added NDP for the donor and the
acceptor sites are the determinant for the ratio [3H]CMP/
[3H]CTP Upon cold CDP addition, this ratio was still found
to be close to 1 In contrast, upon dCDP addition, the
[3H]CMP formation was found to increase with dCDP, while
[3H]CTP remained very low (Fig 3A, lane 5–7 and 3B) This
indicates that dCDP binds preferentially to the donor site
where it is transformed into dCTP We then examined the
effect ofL-3TCDP addition As shown in Fig 3, the amount
of [3H]CMP decreased in a dose-dependent way and
[3H]CTP remained very low We conclude that, in contrast
to dCDP,L-3TCDP was not phosphorylated at the donor
site, but rather that it constitutes a competitor in the reaction
(Fig 3A, lane 8–10 and Fig 3B) Each nucleotide site has a
different specificity, the donor site binding D-nucleotides
only and the acceptor site recognizing UMP, CMP and
L-analogs likeL-3TCMP andL-3TCDP
Discussion
Human UMP-CMP kinase has been recently a subject of
interest for several research groups In 1999, Van Rompay
et al reported the first cloningof the human gene and
characterization of the correspondingprotein, UMP-CMP
kinase [4] The enzyme was expressed as a GST fusion
protein to facilitate its purification Duringthe course of the
present study, Cheng’s laboratory also cloned the human
enzyme and provided a measure of kinetic parameters They
also showed that it was predominantly localized in the
cytoplasm [13] We observed that the enzyme with an
N-terminal His-tagpresents better kinetic performances
than the GST-fusion protein The enzyme was found to be
active in Tris buffer duringa few hours only but
stabiliza-tion by glycerol significantly improves the reproducibility of
data Addition of dithiothreitol in the assay also improved
the measures as previously shown [4,13,23] Under these
conditions, the turnover of human UMP-CMP kinase
(kcat¼ 130 s)1) is the highest of the NMP kinases family
This high activity is particularly striking when compared to
human TMP kinase (kcat¼ 1 s)1) [24,25]
Inhibition by excess of substrate was observed for UMP
and CMP but not for dCMP, AraCMP and L-3TCMP
Previous studies did not report such an inhibition, probably
because the range of concentrations investigated was too
narrow Such a substrate inhibition can be attributed to a
non-productive bindingof CMP and UMP, or to the
products CDP and UDP, forminga dead-end complex It is
correlated to the presence of the 2¢-OH of the sugar in the cis
position The latter is absent in dCMP andL-3TCMP, and
is in trans in the arabinose epimer The nonproductive
bindingof NMP due to the 2¢-OH in cis could occur at the
donor site A similar situation has been observed by X-ray
analysis in the Drosophila deoxynucleoside kinase with an
additional ATP bound to the acceptor site [26] Such an
inhibition is an important parameter to take into account
when the kinase activity is characterized in cellular extracts
Indeed, the kinase specificity profile is used to characterize
the contribution of the cellular kinases in tissues and is often determined usingonly one saturatingconcentration of each substrate Obviously, choosinga high substrate concentra-tion, as one usually does to perform enzyme assay, may provide misleadingresults
The reactivity of both sites of UMP-CMP kinase for
L-3TC mono or diphosphate was studied The acceptor site
is able to bind and to phosphorylate L-3TCMP in the forward reaction (Figs 1B and 2) At the donor site,
L-3TCDP was found to be an inhibitor, while dCDP was
a substrate (Fig 3) ThereforeL-3TCTP cannot be synthes-ized by human UMP-CMP kinase Solvingthe structure
of human UMP-CMP kinase by X-ray crystallography
is currently underway and will help to understand the molecular basis of this substrate specificity
Several enzymes of the nucleoside kinases family have been shown to recognize L-derivatives as substrates, in particular at the acceptor site, includingdeoxycytidine kinase, human mitochondria thymidine kinase 2 and herpes thymidine kinase 1 and 2 [27–29] Only human deoxycyti-dine kinase was previously shown to have a relaxed enantioselectivity at the donor site forL-ATP [30] This is not the case for human UMP-CMP kinase, asL-3TCDP is not a substrate at the donor site Enzymes from virus and phages including HIV reverse transcriptase, HBV DNA polymerase, human DNA primase and T4 DNA ligase have been also reported to use L-NTPs as substrates [31] The three steps of the cellular activation of L-3TC involve deoxycytidine kinase [30], UMP-CMP kinase [13] (data not shown) and phosphoglycerate kinase [14] Most human degradation enzymes like cytidine deaminase present a strict enantioselectivity for D-nucleotides [11,29] that probably contribute to the high stability of the phosphorylated forms
ofL-3TC in cells and to a high cellular level ofL-3TCTP Several laboratories have tried to improve the catalytic properties of herpes virus thymidine kinase and human thymidylate kinase for antiviral drugs An improved herpes virus enzyme in combination with AZT could be used as a suicide enzyme for cancer cells [32] Takinginto account its high efficiency in phosphorylating several nucleotide ana-logs, human UMP-CMP kinase could provide a model for new approaches in suicide enzymes therapies
Acknowledgements
We wish to thank A Karlsson (Karolinska Institute, Stockholm, Sweden) for kind gift of the plasmid coding for GST-fusion UMP-CMP kinase, and our colleagues from the Institut Pasteur for their kind help: M Delarue for modelling L -3TCMP complexed to UMP-CMP kinase, A Haouz for DLS measurements, A Cardona for BETA-Imager counting, C Guerreiro for kind advises about L -3TCMP purification, E Seclaman for helpingcloningthe gene of human UMP-CMP kinase, N Duchange for the kind gift of human cDNA bank and
O Barzu for stimulatingdiscussions This work was supported in part
by a grant from Agence Nationale pour la Recherche contre le SIDA (France) HML was supported by Institut National de la Sante´ et de la Recherche Me´dicale (France) and CP by University of Ferrara (Italy).
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