Pre-steady State of Reaction of Nucleoside Diphosphate Kinase
with Anti-HIV Nucleotides*
Benoit
Schneider
,
Ying Wu
Xu§,
Olivier
Sellam
,
Robert
Sarfati¶,
Joel
Janin§,
Michel
Veron
, and
Dominique
Deville-Bonne
From the
Unité de Régulation Enzymatique
des Activités Cellulaires, CNRS URA 1149 and ¶ Unité
de Chimie Organique, Institut Pasteur, 75724 Paris Cedex 15, and
the § Laboratoire d'Enzymologie et de Biochimie
Structurales, CNRS UPR 9063, 91198 Gif-sur-Yvette, France
 |
ABSTRACT |
The pre-steady-state reaction of
Dictyostelium nucleoside diphosphate (NDP) kinase with
dideoxynucleotide triphosphates (ddNTP) and AZT triphosphate was
studied by quenching of protein fluorescence after manual mixing or by
stopped flow. The fluorescence signal, which is correlated with the
phosphorylation state of the catalytic histidine in the enzyme active
site, decreases upon ddNTP addition according to a monoexponential time
course. The pseudo-first order rate constant was determined for
different concentrations of the various ddNTPs and was found to be
saturable. The data are compatible with a two-step reaction scheme,
where fast association of the enzyme with the dideoxynucleotide is
followed by a rate-limiting phosphorylation step. The rate constants
and dissociation equilibrium constants determined for each
dideoxynucleotide were correlated with the steady-state kinetic
parameters measured in the enzymatic assay in the presence of the two
substrates. It is shown that ddNTPs and AZT triphosphate are poor
substrates for NDP kinase with a rate of phosphate transfer of 0.02 to
3.5 s
1 and a KS of 1-5
mM. The equilibrium dissociation constants for ADP, GDP,
ddADP, and ddGDP were also determined by fluorescence titration of a
mutant F64W NDP kinase, where the introduction of a tryptophan at the
nucleotide binding site provides a direct spectroscopic probe. The lack
of the 3'-OH in ddNTP causes a 10-fold increase in
KD. Contrary to "natural" NTPs, NDP kinase discriminates between various ddNTPs, with ddGTP the more efficient and
ddCTP the least efficient substrate within a range of 100 in
kcat values.
 |
INTRODUCTION |
Nucleoside analogues like 3'-deoxy-3'-azidothymidine
(AZT)1 and dideoxynucleosides
(ddN) are widely used as antiviral drugs, particularly in the
multitherapy protocols now used in the treatment of AIDS. These drugs
are targeted at the HIV reverse transcriptase as the lack of the 3'-OH
required for the 3'-5' phosphodiester bond formation during DNA
elongation blocks viral DNA synthesis. To exert their antiviral
activity, the nucleoside analogues must be phosphorylated into
triphosphates derivatives by cellular kinases. Although AZT, ddT, and
ddC are structurally related, they show different patterns of
intracellular phosphorylation (1). The synthesis of mono- and
diphospho-derivatives involves kinases specific for either purines (for
example deoxyguanosine kinase) or pyrimidines (for example
deoxycytidine kinase or thymidine kinase). In all cases, the last step
in the pathway leading to the triphospho-derivative is catalyzed by
nucleoside diphosphate (NDP) kinase (EC 2.7.4.6), which has little
specificity toward the nucleobase (2).
NDP kinase phosphorylates all nucleoside diphosphates into
triphosphates using ATP as the major phosphate donor. The reaction involves the formation of a phosphorylated intermediate according to a
ping-pong bi-bi mechanism following the scheme below, where E-P is the phosphorylated intermediate on the catalytic
histidine (3).
In recent years, genes encoding NDP kinase have been cloned from a
number of prokaryotic and eukaryotic organisms (4, 5). NDP kinases are
made up from 17-kDa subunits with highly conserved sequences. The x-ray
structures of NDP kinases from several species have also been
determined at high resolution (5-8), and these studies have shown that
both the subunit fold and the active site are remarkably conserved.
Solving the NDP kinase structure in the presence of dTDP (9), ADP (10),
and GDP (11) was an important step toward an understanding of the
phosphate transfer mechanism. The nucleotide binding site is very
different from other known nucleotide-binding proteins, with the base
stacking on a phenylalanine near the protein surface without polar
interactions with the protein side chains. The ribose and phosphate
moieties are located deeper inside the active site, forming numerous
bonds with a Mg2+ ion and protein side chains. The
nucleotide conformation is original, with a hydrogen bond between the
3'-OH of the sugar and the
-phosphate. In addition, the 3'-OH
accepts H-bonds from Lys16 and Asn119 (in this
report, we use the numbering of Dictyostelium NDP kinase). The catalytic His122 points its N
toward the phosphate,
a well defined water molecule bridging it to the
-phosphate oxygen
in the ADP complex, at the presumed position of ATP
-phosphate.
A precise model for the reaction product and the transition state has
been proposed, based on the crystal structures of the enzyme
phosphorylated by phosphoramidate (12) and of the ternary complex with
ADP and AlF3 (13). This model is also supported by the
results of a large series of substitutions of conserved residues
of the active site (14). NDP kinase has a very high turnover with
kcat around 1000 s
1 for
"natural" ribo- or deoxyribonucleotides. Comparing the
structures of free enzyme and enzyme complexed with nucleotide
diphosphates demonstrates that the change of conformation upon
nucleotide binding is minimal. The movement is limited to the
A/
2 helices hairpin, which forms one side
of the nucleotide binding site (8-10). Dictyostelium NDP
kinase has a single tryptophan (Trp137), located at the
proximity of the active site. We have shown previously that its
fluorescence could be used as a probe for the phosphorylation state of
the catalytic histidine (15).
We have recently reported that the diphospho-derivatives of some
nucleotide analogues used as antiviral drugs are poor substrates for
NDP kinase, as measured by the global enzymatic assay (16). Thus, when
the diphospho- and triphospho-derivatives of azidothymidine, dideoxyadenosine, or dideoxythymidine are used as substrates, the rate
of phosphate transfer is 102 to 104 times less
than for natural nucleotides.
In this study, we present pre-steady-state kinetic experiments to
investigate the phosphorylation of the enzyme by antiviral ddNTPs. Both
stopped-flow and conventional techniques were used to measure
fluorescence changes on a time scale ranging from milliseconds to
several minutes. Using a mutant NDP kinase (F64W) in which an
additional tryptophan was placed near the nucleotide binding site, we
also measured the affinity constants of nucleotides and their
analogues.
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EXPERIMENTAL PROCEDURES |
Materials--
ATP, ADP, CDP, GDP, dTDP, lactate dehydrogenase,
and pyruvate kinase were from Boehringer Mannheim, and
dideoxynucleotide triphosphates from Amersham Pharmacia Biotech or from
Boehringer Mannheim. [14C]ADP (57 mCi/mmol) was from NEN
Life Science Products. The synthesis of phosphoderivatives of AZT and
of ddNDP has been described previously (16).
Site-directed Mutagenesis--
The mutation F64W in
Dictyostelium NDP kinase was made by site-directed
mutagenesis according to Kunkel (17), using the oligonucleotide
5'-GAAAGACCATGGTTCGGTGGTT-3'. Altered bases as
compared with the wild type sequence are bold and underlined. The
mutation was verified by DNA sequencing.
Enzyme Purification--
Wild type and mutant
Dictyostelium NDP kinase were overexpressed in
Escherichia coli (XL1-Blue) using plasmid pndk as
described (4) with small modifications. The cell extract was loaded at pH 8.4 onto DEAE-Sephacel which retained only E. coli NDP
kinase (14) and the flow-through was adsorbed on Blue-Sepharose
(Amersham Pharmacia Biotech) at pH 7.5. After washing with Tris buffer, the enzyme was eluted by a NaCl gradient (0-1.5 M) in 50 mM Tris-HCl, pH 7.5. After dialysis, the protein was
concentrated with an Amicon ultrafiltration cell, equilibrated in 50 mM Tris-HCl, pH 7.5, and stored frozen at
20 °C.
Protein concentration was determined using an absorbance coefficient of
A280 = 0.55 for a 1 mg/ml solution. Mutant
F64W NDP kinase was purified according to the same procedure. The
absorption coefficient of F64W mutated NDP kinase was estimated to
A280 = 0.85 for a 1 mg/ml solution according to Gill equation for a folded protein in water (18). All proteins were
purified to homogeneity as judged by SDS-polyacrylamide gel electrophoresis. Enzyme concentration was expressed as concentration of
17-kDa subunits.
The phosphorylated enzyme was prepared as described previously (15);
the enzyme that had been preincubated in T buffer (50 mM
Tris-HCl, pH 7.5, containing 5 mM MgCl2 and 75 mM KCl) with a saturating amount of ATP was made free of
nucleotides by gel filtration on Sephadex G-25. The concentration of
the phosphorylated enzyme as well as the absence of nucleotides were
checked from the absorbance spectrum of the protein. The stoichiometry
of phosphorylation was determined in a parallel experiment using
[
-32P]ATP, as described in Ref. 15. The phosphorylated
enzyme was then kept at 4 °C and used within 3 h after
synthesis.
Activity Assays--
Two different assays were used to measure
NDP kinase activity. In the first assay, activity of NDP kinase was
measured at 20 °C with ATP and dTDP as substrates using coupled
enzymes (lactate dehydrogenase and pyruvate kinase) as described
previously (19). The second assay was used when small reaction volumes
(a few microliters) were needed, in particular with radioactive
substrates (16). When ddNTPs were tested as phosphate donors,
[14C]ADP was used as an acceptor. The initial rate of the
reaction was determined at 20 °C in the presence of a constant
amount of [14C]ADP (0.1 mM) during 2, 4, and
6 min. After separation of the radioactive nucleotides by thin layer
chromatography on PEI-cellulose (Macherey-Nagel, Germany), their
radioactivity was quantified using a PhosphorImager (Molecular
Dynamics).
The ratio of apparent kcat/apparent
Km, measured at a constant concentration of the
other substrate is equal to the true value of
kcat/Km for a ping-pong
enzyme. It is a useful parameter when comparing natural substrates to
nucleotides analogs. The nonlinear least-squares fit of the data was
performed using Kaleidagraph (Abelbeck Software). Unless otherwise
indicated, this software was also used for all of the fittings
described below.
Binding Studies--
The affinity of NDP for NDP kinase was
measured by following the variation of intrinsic fluorescence of the
mutant F64W enzyme upon nucleotide binding. All fluorescence
measurements were performed at 20 °C in T buffer on a Photon
Technology International (PTI) spectrofluorometer (QuantamasterTM).
Successive aliquots of the nucleotide were added to the enzyme solution
(2 µM), and the fluorescence was measured at 340 nm with
excitation at 295 nm for ADP and ddADP (2-nm excitation slit and 2-nm
emission slit), or at 304 nm in the case of the other nucleotides
(emission slit was then 4 nm). Experimental binding curves were fitted
to a hyperbolic ligand-protein curve after correction for dilution. The
inner filter effect was found to be negligible.
Slow Kinetics Experiments--
Slow kinetics experiments
were performed at 20 °C on a PTI spectrofluorometer
(QuantamasterTM), sampled with continuous stirring. The reaction of
wild type NDP kinase with NTP analogues was initiated by addition of
the nucleotide (less than 25 µl) to 1 ml of a 0.85 µM
enzyme solution in buffer T in a Hellma cell with four optical windows.
In a similar manner, the reaction of phosphorylated enzyme (1 µM) with NDP analogues was monitored in a 1-ml optical
cell after addition of the substrate in a volume less than 25 µl. The time required for manual mixing was less than 15 s. The tryptophan fluorescence of NDP kinase was monitored for up to 10 min at 340 nm,
with excitation at 295 nm or 304 nm as above. The fitted curves were
found to correspond to a single exponential progress, either decreasing
for phosphate transfer to the enzyme or increasing in the case of
phosphate transfer to the NDP analogues. The pseudo first-order rate
constants (kobs) were determined as a function of substrate concentration (S), with [S] in excess to
[E], the enzyme concentration.
Stopped-flow Kinetic Experiments--
Stopped-flow kinetic
experiments were performed at 20 °C in buffer T with an Applied
Photophysics SX.18MV microvolume stopped-flow reaction analyzer
equipped with a high intensity xenon lamp. The excitation wavelength
was 296 nm (for ATP and ddATP measurements) or 305 nm (for all the
other nucleotides), with a 2-mm excitation slit and a 320-nm cutoff
filter at the emission. Mixing was achieved in less than 2 ms. After
mixing NDP kinase (0.85 µM, final concentration) and
ddNTP or ddNDP (25-3000 µM, final concentration), the
intrinsic protein fluorescence was recorded for 10 to 50 s. In
each experiment 400 pairs of data were recorded, and the data from
three or four experiments under identical conditions were averaged and
then fitted to a number of nonlinear analytical equations using the Pro/kineticist software provided by Applied Photophysics. The fitted
curves correspond to a single exponential.
 |
RESULTS |
Changes in Fluorescence Associated with Phosphorylation of NDP
Kinase by Antiviral Nucleotide Analogues--
We have shown previously
that the fluorescence of the single tryptophan in
Dictyostelium NDP kinase is quenched upon phosphorylation of
the enzyme by ATP (15). To test whether the interaction with ddNTP also
results in a change of fluorescence, steady-state emission spectra
(
exc = 295 nm) were collected in presence of ddATP or ddADP at saturating concentrations, and compared with the spectrum of
the native enzyme. A strong decrease (20%) in fluorescence intensity
near 320-340 nm was observed in the presence of ddATP. In contrast,
the fluorescence was insensitive (less than 3% decrease) to ddADP
binding, as reported previously for ADP. Quenching (at least 10%) was
also observed with other natural NTPs or ddNTPs (
exc = 304 nm) and was likely due to the phosphorylation of the catalytic
histidine. Conversely, the fluorescence signal of the phosphorylated
enzyme was enhanced upon ddADP addition as the phosphate was
transferred from the protein back to the nucleotide. This provided a
convenient method to monitor histidine phosphorylation at different
ddNTP/ddNDP ratios and to derive the equilibrium constants. As shown in
Fig. 1, the equilibrium constant was the same for the ddTTP/ddTDP pair (Keq = 0.13 ± 0.02) and for the AZTTP/AZTDP pair. It did not differ significantly
from natural nucleotides. A value of Keq = 0.19 ± 0.02 was obtained previously for the ATP/ADP pair by
fluorescence titration (15). Applying the Haldane relationship to
steady-state kinetic parameters yielded the same value. We conclude
that the absence of 3'-OH or its replacement with an azido group in the
analogue has no significant effect on the phosphorylation equilibrium
between the enzyme and the nucleotides.

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Fig. 1.
Interaction at the equilibrium of NDP kinase
and ddTTP/ddTDP or AZTTP/AZTDP. Wild type NDP kinase (1 µM) in buffer T was incubated at the equilibrium at
20 °C with 50 µM nucleotides in total amount and in
varying [NDP]/[NTP] ratio: ddTXP, ; AZT-XP, . Data were
fitted with the following equation: Keq = ([E-P] [NDP])/([E] [NTP]) modified to the
following formulation: F = ( Fmax·r)/(r + Keq), where r = [NDP]/[NTP],
Fmax the maximum variation in fluorescence,
and F the variation observed at a given ratio
r. Fitting is shown as solid line for AZT-XP
only. The value for Keq is 0.13 ± 0.02 in
both cases. The saturation curve appears here as a sigmoid due to the
logarithmic representation of the x axis.
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Steady-state Kinetic Parameters of NDP Kinase with
ddNTP--
Steady-state kinetic parameters were first measured in the
phosphate exchange reaction between ddNTP and [14C]ADP
(reactions A and B, shown in Scheme I). Fig.
2 shows initial rate data for the native
enzyme for ddGTP at various concentrations and the inset
shows the comparison of the four ddNTPs at a given concentration. A
constant concentration of ADP (0.1 mM), corresponding to
twice the KmADP (20), was used.
The data were adjusted with the Michaelis equation. Table
I summarizes the kinetic constants
(apparent Km and apparent
Vmax) determined for ddATP, ddGTP, ddTTP, and
ddCTP used as phosphate donors.

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Fig. 2.
Measure of steady-state kinetics parameters
of Dictyostelium NDP kinase. Initial velocities were
determined in the presence of 0.1 mM [14C]ADP
from time course for phosphate transfer at 2, 4, and 6 min with ddGTP
as phosphate donors. The apparent Vmax and the
apparent Km were obtained by fitting the data with
the Michaelis equation using Kaleidagraph. Inset, time
course of the reaction at 25 mM ddNTP as phosphate donors:
ddGTP, ; ddATP, ; ddTTP, ; ddCTP, .
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Table I
Comparison of steady-state parameters of NDP kinase for nucleoside
triphosphates with rates and equilibrium constants determined in
pre-steady-state experiments
Stopped-flow parameters values correspond to data from Fig.
4A. Hand-mixing parameters correspond to data from Fig.
4B. All the experiments were performed at 20 °C. The
number in parentheses corresponds to the number of independent
measurements. ND, not determined.
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The lack of the 3'-OH in ddNTP dramatically affected the catalytic
activity. An increase by a factor of 10 in apparent
Km and a decrease of 500-10,000 in apparent
kcat were observed when comparing kinetic
parameters of ddNTPs with those of ATP (20). Under the conditions used,
the kinetic parameters of natural nucleotides were very similar
(results not shown). In contrast, the kinetic constants of
dideoxynucleotides varied strongly with the ddNTP used. In particular,
ddCTP showed a very low value of
kcat/Km, with a drop of more
than 104 as compared with CTP.
Pre-steady-state Kinetics of NDP Kinase Phosphorylation by
ddNTPs--
The fluorescence signal allowed us to monitor the time
course of the phosphate transfer reactions between NDP kinase and
nucleotides. However, the time-dependent change of the
fluorescence was too fast to be observed when 1 µM enzyme
was reacted with 100 µM ATP. This is due to the fact that
the time response of the stopped flow is slow compared with the
kcat of the enzyme (~600 s
1 at
20 °C). In contrast, when the enzyme was reacted with ddATP under
the same conditions, a time-dependent quenching of the
enzyme fluorescence was observed in the second-to-minute time scale
(Fig. 3A), with no observable
lag. For all concentrations of ddATP tested, a single exponential decay
was observed, characterized by a pseudo-first order rate constant
kobs. The signal amplitude was constant,
corresponding to a complete phosphorylation of the enzyme. The
pseudo-first order rate constant of the phosphorylation reaction is
shown in Fig. 4A as a function
of [ddATP] in the 0.1-3 mM range. The observed rate
constant (kobs) increased linearly for [ddATP] < 0.2 mM before reaching a plateau. These data were best
adjusted to the equation of a saturation curve with a maximum rate
constant of 1 s
1 at saturating ddATP and an apparent
equilibrium dissociation constant KS of 0.75 mM (Fig. 4A and Table I).

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Fig. 3.
Kinetics of NDP kinase phosphorylation by
ddATP. A, 0.85 µM (final monomer
concentration) NDP kinase in buffer T at 20 °C was rapidly mixed
with 0.1-3 mM ddATP (final concentration) in the same
buffer, and the decrease in fluorescence was monitored with time on the
stopped-flow device. Representative traces are shown for 100 (a), 200 (b), and 400 (c)
µM ddATP. Each reaction was monitored during 200 s,
and the data are plotted on this scale for clarity. The solid
lines represent the best fit of each curve to a monoexponential.
B, residuals for curve b are shown under the
fit.
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Fig. 4.
Pre-steady-state kinetics of NDP kinase
phosphorylation with ddNTP: concentration dependence of
kobs for phosphorylation on [ddNTP].
A, stopped-flow experiments with ddGTP ( ), ddTTP ( ),
ddATP ( ), and ddCTP ( ). The pseudo-first order rate constant for
the reaction is hyperbolically dependent on ddNTP concentration. The
solid lines are best fits of the data to an hyperbolic
saturation curve according to Reaction 1. The values of apparent
equilibrium dissociation constants (KS) and maximum
pseudo first-order rate-constant are given on Table I. B,
hand mixing experiments with ddGTP ( ), ddTTP ( ), ddATP ( ),
ddCTP ( ), as well as AZTTP ( ). The linear fit indicates that data
can be analyzed as a second order reaction, with an apparent constant
given as kobs = k+2/KS in Table I.
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Similar monoexponential decays were found when reacting the enzyme with
ddGTP, ddTTP, and ddCTP. The pseudo-first order rate constants varied
with [ddNTP]. The corresponding parameters (Table I) showed
noticeable differences between nucleotides; thus, ddGTP appears to be
the best substrate, ddATP and ddTTP being slightly less efficient.
ddCTP is a very poor substrate for NDP kinase, which was nearly
inactive with ddCTP in the 0.1-3 mM range. Therefore, the
kinetic parameters for ddCTP could not be determined precisely.
Fig. 4B shows a similar dependence of the pseudo-first
order rate constant when the reaction was initiated by hand mixing. In
this case, only the linear part of the previous saturation curves were
measured. The slope had the dimension of a bimolecular association rate
constant and was sufficient to characterize the interaction of NDP
kinase with low concentrations of NTP analogues. The values for these
slopes are given as k+2/KS (Table I). In all cases (stopped-flow or hand mixing experiments), the
very low intercepts with the vertical axis (~0.1 s
1 or
less) indicate that the phosphorylated intermediate generated during
the reaction was stable in the absence of a NDP acceptor substrate.
Pre-steady-state Kinetics of NDP Kinase Dephosphorylation with
ddNDP--
The dephosphorylation of the phosphorylated enzyme
(E~P) by ddTDP and by AZTDP (reaction B, Scheme I) also
displayed a monoexponential time course in the minute range (Fig.
5). The apparent time constant (apparent
kdephos) for ddTDP was about 5 times that of the
apparent kobs for phosphorylation by ddTTP
(reaction A, Scheme I) at the same nucleotide concentration.

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Fig. 5.
Pre-steady-state kinetics of
dephosphorylation of NDP kinase with ddTDP and AZTDP (hand
mixing). Concentration dependence of the pseudo-first order
constant kobs for the dephosphorylation as a
function of ddTDP ( ) and AZTDP ( ).The apparent second order
constant is 4500 M 1 × s 1 for
ddTDP and 1500 M 1 × s 1 for
AZTDP.
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Affinity of NDP Kinase Kinase for Nucleotide Diphosphates--
In
order to follow the binding of the nucleotide, we designed a specific
mutant protein with a tryptophan inserted in position 64, replacing
Phe64, which stacks with the nucleobase in the crystal
structure (9, 10). Purified F64W mutant NDP kinase showed essentially
identical kinetic parameters to the wild type enzyme (Table
II) and was stable up to 4.5 M urea. The intrinsic fluorescence of F64W NDP kinase (at
an excitation wavelength of 295 nm, at 20 °C, pH = 7.5) is
primarily due to its two tryptophans in position 64 and 137. The
fluorescence of the F64W mutant enzyme decreased by 10% upon addition
of a saturating amount of ADP (Fig. 6,
inset) whereas wild type enzyme fluorescence was not
affected (15).
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Table II
Effect of Phe64 mutation on the kinetic parameters of
Dictyostelium NDP kinase
The catalytic rate constants (kcat) and Michaelis
constants (Km) were obtained from initial rate
studies performed at a constant ratio of nucleotide concentrations
[dTDP]/[ATP] = 0.05 and 0.1 with [ATP] varying from 0.2 to 2 mM (25). While there are 6 subunits per molecule, turnover
numbers are expressed per subunit (or per active site). The specific
activity is expressed in units/mg (1 unit is the amount of enzyme
catalyzing the transfer of 1 µmol of phosphate/min). Each value is
the mean of three independent determinations for wild type and 2 for
the mutant NDP kinase.
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Fig. 6.
Binding curves of F64W NDP kinase with ADP
and ddADP. Fluorescence change of F64W NDP kinase (2 µM) upon binding of ADP ( ) or ddADP ( ) at 20 °C
in buffer T, pH = 7.5. The solid lines are best fits of
the data to an hyperbolic saturation curve with a KD
of 25 ± 5 µM for ADP and 220 ± 40 µM for ddADP (Table III). Inset, fluorescence
emission spectra of F64W NDP kinase in the absence and in the presence
of ADP. The emission spectra of F64W NDP kinase (2 µM in
subunits) in buffer T is modified by the addition of 0.5 mM
ADP. Spectra are corrected by the PTI procedure.
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We took advantage of this variation in intrinsic fluorescence to
determine the affinity of nucleotide diphosphates for NDP kinase under
equilibrium conditions. Fig. 6 shows a typical binding curve, where the
fluorescence decreases as a function of [ADP]. The values of the
dissociation constant KD at equilibrium for various
nucleotides calculated from such binding curves are shown in Table
III.
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Table III
Dissociation constants of F64W NDP kinase for selected nucleoside
diphosphates
Titration of F64W enzyme with natural NDP, their corresponding
dideoxynucleotides, as well as with AZTDP was monitored by the
quenching of enzyme fluorescence (excitation wavelength of 295 nm
(adenosine derivatives) or 304 nm and emission wavelength of 340 nm). The number in parentheses indicates the number of independent
measurements.
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Dideoxynucleotide diphosphates displayed lower affinity for NDP kinase
than the natural substrates (Fig. 6). The KD values
were, respectively, 25 µM and 220 µM for
ADP and ddADP and 14 µM and 120 µM for GDP
and ddGDP (Table III). The absence of a 3'-OH resulted in the loss of
affinity equivalent to 1.3 kcal/mol of binding free energy. However,
stopped-flow experiments performed with 1 µM of F64W NDP
kinase and 100 µM ddADP failed to detect a
time-dependent variation in fluorescence, indicating that
the association reaction was completed within the mixing time and,
therefore, that the bimolecular rate constant was at least 2 × 106 M
1 × s
1.
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DISCUSSION |
In this work, we have conducted steady-state and pre-steady-state
experiments to study the interaction of NDP kinase with antiviral
dideoxynucleotide triphosphates as substrates. The enzyme from the
slime mold Dictyostelium discoideum is a 102-kDa hexamer. It
is highly homologous to both isoforms of the human enzyme (57% sequence identity), and has a very similar three-dimensional structure, especially at the active site (8-10) (Fig.
7). Conclusions drawn from this study
should therefore be applicable to the human enzymes. The structure of
AZT-DP complexed with a point mutant of Dictyostelium NDP
kinase was recently resolved (21). It shows that the analogue binds at
the same site and in the same orientation as the natural substrate
dTDP. As could be expected, the phosphorylation equilibrium between the
enzyme and its substrates is unaffected by the absence of the 3'-OH in
dideoxynucleotides or its substitution in AZT derivatives. However, the
analogues are very poor substrates (16), and we have found in the
present study that the rate of phosphate transfer was strongly reduced
in pre-steady-state experiments when either the histidine
phosphorylation or the dephosphorylation step was followed as a
function of time and substrate concentration. In
Dictyostelium NDP kinase, the fluorescence of
Trp137 is the signal which we use to monitor the phosphate
transfer step in the kinetics, as it is sensitive to the state of
histidine phosphorylation, but not to substrate binding.

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Fig. 7.
Nucleotide binding site. Figure is a
stereoview of the NDP kinase active site with bound dTDP (adapted from
Ref. 9). The thymine base is stacked between Phe64 and
Val116 at the entrance of the active site and points down
toward outside the protein. The phosphates carry a Mg2+ ion
and point toward the active His122 on top. The single
Trp137 is located near the catalytic His122.
The 3'-OH of the natural nucleotide makes hydrogen bonds with residues
Lys16 and Asn119 and with the phosphate.
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Our kinetic data are compatible with the following reaction.
When studying the forward reaction, the concentration of the ddNDP
product remains very low and the product binding reaction can be
neglected. Under these circumstances, the
[E-P·ddNDP]/[E-P] ratio must be less than
k+2/k'
1 and therefore
much smaller than 1, given the very low rate of phosphorylation
observed with the analogues. Then, the mechanism of phosphorylation by ddNTP simplifies to Reaction 2.
When studying dephosphorylation by ddNDP in the absence of ddNTP,
the same argument leads to writing the reaction mechanism as shown in
Reaction 3.
The bimolecular steps are expected to be fast in both directions,
and in neither type of experiment are they directly observable in the
kinetics. For Reaction 2, and in large excess of the ddNTP ligand, the
rate of the single step that is observed should be as shown below (22,
23).
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(Eq. 1)
|
The observed rate of phosphorylation should increase with
[ddNTP] to reach a plateau value equal to k+2,
the true rate of histidine phosphorylation, with half-saturation
occurring for [ddNTP] = k
1/k+1
=KS, the equilibrium dissociation constant of the
nucleotide substrate. At low [ddNTP], kobs is expected to increase linearly with [ddNTP] with an apparent
bimolecular rate constant that is
k+2/KS.
Equation 1 was used to fit the curves in Fig. 4 (A and
B), yielding rate and dissociation constants listed in Table
I. Whereas the two purine dideoxynucleotides have similar dissociation
constants KS
1 mM, the rate of
phosphorylation is larger for ddGTP (k+2 = 3.5 s
1) than for ddATP (1 s
1). On the other
hand, pyrimidine dideoxynucleotides are relatively poorer substrates
for NDP kinase phosphorylation. ddTDP has k+2
2.5 s
1 and KS
5 mM, too large a value for saturation to be reached under
our experimental concentrations. With ddCTP, the catalytic efficiency
is so poor that neither KS nor the rate of phosphate
transfer could safely be determined in stopped-flow experiments at
nucleotide concentrations up to 3 mM.
Because the affinity of NDP kinase for its natural nucleotide
substrates could not be determined in the same way, we resorted to
designing a mutant where a tryptophan replaces Phe64 at the
base binding site (Fig. 7). The substitution provides a spectroscopic
signal that monitors ligand binding. The stability and steady-state
catalytic properties of the F64W NDP kinase given in Table II are very
similar to those of the wild type enzyme. Equilibrium dissociation
constants for E·NDP dead-end complexes were determined by
fluorescence titration for the natural nucleotides and their analogues.
Values listed in Table III show that the absence of the 3'-OH in the
dideoxy analogues raises the dissociation constant by a factor of about
10 in purine nucleotides. This same ratio is also seen in steady-state
parameters, Km values being
10 times larger for
ddNTP than for NTP substrates (16). Differences between natural
nucleotides are apparent in Table III, with ADP and GDP having similar
affinities that are significantly better than for dTDP and especially
CDP. The trends are the same for KS values obtained
for the ddNTPs from the analysis of pre-steady-state kinetics
above.
Although the dephosphorylation reaction was not studied in the same
details, our data suggest that it obeys similar rules. Phosphate
transfer from the phospho-enzyme to a dideoxynucleoside diphosphate
substrate is slow and rate-limiting compared with substrate binding.
Because saturation of the observed rate of dephosphorylation was not
achieved at substrate concentrations used for manual mixing experiments
(Fig. 5), only the apparent bimolecular rate constant
k
2/KS can be derived from
these data. For ddTTP,
k
2/KS = 4500 M
1 s
1, which exceeds by a ratio
of
5 the corresponding value of
k+2/KS values found for ddTTP
in Table I. According to the Haldane equation, this ratio should be
equal to the equilibrium constant Keq for phosphorylation, which we find to be 1/0.13
7 by direct
measurement (Fig. 1). The two determinations of
Keq are completely independent, and their
agreement strongly supports our interpretation of the kinetic data.
An early study by Wälinder et al., in 1969, investigated the phosphorylation of bovine NDP kinase by
[
-32P]ATP using a rapid mixing technique (24). The
pseudo-first order rate constants for phosphorylation by ATP and for
dephosphorylation by dGDP exceeded the turnover number of the overall
reaction, indicating that the phosphoenzyme could be an intermediate in the NDP kinase reaction. With these substrates, both steps are fast
and, in the case of the Dictyostelium enzyme where
kcat is on the order of 1000 s
1,
they are completed in less than a millisecond, too fast for stopped-flow studies. With less efficient substrates such as the antiviral analogues studied here, the turnover rate constant drops to
1-2 s
1. Phosphorylation by ddNTP in one direction and
dephosphorylation by ddNDP in the other direction are slow and
rate-limiting in the overall reaction. Accordingly, the rates of
phosphorylation k+2 derived from the analysis of
pre-steady-state data are in very good agreement with steady-state
kcat values measured with the same nucleotide
analogues as substrates (Table I).
Our results indicate that the absence of the 3'-OH in the analogues
result in a 10-fold increase in the dissociation constant and in a
300-5000 decrease in the rate of phosphate transfer, resulting in a
factor 3 × 103 to 5 × 104 in
catalytic efficiency. The 3'-OH of the nucleotide sugar is involved in
a hydrogen bond network with Asn119, Lys16 on
the protein, and also with the oxygen that bridges the
- and
-phosphates of the nucleotide itself (Fig. 7). Removing the Asn119 or Lys16 side chains results in mutant
NDP kinases that display a much less dramatic decrease in catalytic
efficiency than when the 3'-OH is removed;
kcat/Km drops by a factor of
10 in the N119A mutant (21) and by a factor of 200 in the K16A
mutant.2 The loss of the
internal hydrogen bond between the 3'-OH and the bridging phosphate
oxygen in ddNTP is likely to be the major reason for the low activity
of the enzyme on dideoxy- or AZT derivative substrates. Additional
differences are observed between the nucleotide analogues themselves,
with ddCTP being the poorest substrate of all, but these differences
have no obvious interpretation at present.
 |
ACKNOWLEDGEMENTS |
We thank D. Pantaloni for making the
stopped-flow apparatus available to us, I. Lascu, M. Desmadril, A. Chaffotte, and J. Stock for helpful discussions, and Manuel Babolat for
expert technical assistance.
 |
FOOTNOTES |
*
This work was supported by grants from the Agence Nationale
de la Recherche contre le SIDA (ANRS AC 14) and from the Ligue contre
le Cancer (Comité de Paris).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Unité de
Régulation Enzymatique des Activités Cellulaires, Institut
Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France. Tel.:
33-1-40-61-35-35; Fax: 33-1-45-68-83-99; E-mail:
ddeville{at}pasteur.fr.
1
The abbreviations used are: AZT,
3'-deoxy-3'-azidothymidine; ddNDP, 2',3'-dideoxynucleoside diphosphate;
ddNTP, 2',3'-dideoxy-nucleoside triphosphate; HIV, human
immunodeficiency virus.
2
B. Schneider, M. Véron, and D. Deville-Bonne, unpublished results.
 |
REFERENCES |
-
Balzarini, J.,
Herdewijn, P.,
and De Clercq, E.
(1989)
J. Biol. Chem.
264,
6127-6133[Abstract/Free Full Text]
-
Parks, R. E. J.,
and Agarwal, R. P.
(1973)
in
The Enzymes (Boyer, P.D., ed), Vol. 8, pp. 307-334, Academic Press, New York
-
Mourad, N.,
and Parks, R. E., Jr.
(1966)
J. Biol. Chem.
241,
3838-3844[Abstract/Free Full Text]
-
Lacombe, M.-L.,
Wallet, V.,
Troll, H.,
and Véron, M.
(1990)
J. Biol. Chem.
265,
10012-10018[Abstract/Free Full Text]
-
Dumas, C.,
Lascu, I.,
Moréra, S.,
Glaser, P.,
Fourme, R.,
Wallet, V.,
Lacombe, M.-L.,
Véron, M.,
and Janin, J.
(1992)
EMBO J.
11,
3203-3208[Abstract]
-
Chiadmi, M.,
Moréra, S.,
Lascu, I.,
Dumas, C.,
LeBras, G.,
Véron, M.,
and Janin, J.
(1993)
Structure
1,
283-293[Medline]
[Order article via Infotrieve]
-
Williams, R. L.,
Oren, D. A.,
Munoz-Dorado, J.,
Inouye, S.,
Inouye, M.,
and Arnold, E.
(1993)
J. Mol. Biol.
234,
1230-1247[CrossRef][Medline]
[Order article via Infotrieve]
-
Webb, P. A.,
Perisic, O.,
Mendola, C. E.,
Backer, J. M.,
and Williams, R. L.
(1995)
J. Mol. Biol.
251,
574-587[CrossRef][Medline]
[Order article via Infotrieve]
-
Cherfils, J.,
Moréra, S.,
Lascu, I.,
Véron, M.,
and Janin, J.
(1994)
Biochemistry
33,
9062-9069[Medline]
[Order article via Infotrieve]
-
Moréra, S.,
Lascu, I.,
Dumas, C.,
LeBras, G.,
Briozzo, P.,
Véron, M.,
and Janin, J.
(1994)
Biochemistry
33,
459-467[Medline]
[Order article via Infotrieve]
-
Moréra, S.,
Lacombe, M.-L.,
Xu, Y.,
LeBras, G.,
and Janin, J.
(1995)
Structure
3,
1307-1314[Medline]
[Order article via Infotrieve]
-
Moréra, S.,
Chiadmi, M.,
LeBras, G.,
Lascu, I.,
and Janin, J.
(1995)
Biochemistry
34,
11062-11070[Medline]
[Order article via Infotrieve]
-
Xu, Y.,
Moréra, S.,
Janin, J.,
and Cherfils, J.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
3579-3583[Abstract/Free Full Text]
-
Tepper, A.,
Dammann, H.,
Bominaar, A. A.,
and Véron, M.
(1994)
J. Biol. Chem.
269,
32175-32180[Abstract/Free Full Text]
-
Deville-Bonne, D.,
Sellam, O.,
Merola, F.,
Lascu, I.,
Desmadril, M.,
and Véron, M.
(1996)
Biochemistry
35,
14643-14650[CrossRef][Medline]
[Order article via Infotrieve]
-
Bourdais, J.,
Biondi, R.,
Sarfati, S.,
Guerreiro, C.,
Lascu, I.,
Janin, J.,
and Véron, M.
(1996)
J. Biol. Chem.
271,
7887-7890[Abstract/Free Full Text]
-
Kunkel, T. A.
(1985)
Proc. Natl. Acad. Sci. U. S. A.
82,
488-492[Abstract]
-
Gill, S. C.,
and von Hippel, P. H.
(1989)
Anal. Biochem.
182,
319-326[Medline]
[Order article via Infotrieve]
-
Lascu, I.,
Chaffotte, A.,
Limbourg-Bouchon, B.,
and Véron, M.
(1992)
J. Biol. Chem.
267,
12775-12781[Abstract/Free Full Text]
-
Lascu, I.,
Deville-Bonne, D.,
Glazer, P.,
and Véron, M.
(1993)
J. Biol. Chem.
268,
20268-20275[Abstract/Free Full Text]; Correction (1994) J. Biol. Chem. 269, 7046
-
Xu, Y.,
Sellam, O.,
Moréra, S.,
Sarfati, S.,
Biondi, R.,
Véron, M.,
and Janin, J.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
7162-7165[Abstract/Free Full Text]
-
Johnson, K.
(1992)
in
The Enzymes (Sigman, D. S., ed), Vol. XX, pp. 2-62, Academic Press, New York
-
Bernasconi, C. F.
(1976)
Relaxation Kinetics, pp. 20-39, Academic Press, New York
-
Wälinder, O.,
Zetterqvist, Ö.,
and Engström, L.
(1969)
J. Biol. Chem.
244,
1060-1064[Medline]
[Order article via Infotrieve]
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.