Pre-steady State of Reaction of Nucleoside Diphosphate Kinase with Anti-HIV Nucleotides*

Benoit SchneiderDagger , Ying Wu Xu§, Olivier SellamDagger , Robert Sarfati, Joel Janin§, Michel VeronDagger , and Dominique Deville-BonneDagger parallel

From the Dagger  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
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Abstract
Introduction
Procedures
Results
Discussion
References

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
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Abstract
Introduction
Procedures
Results
Discussion
References

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).
<UP>         N</UP><SUB>1</SUB><UP>TP</UP>+E ↔ <UP>N<SUB>1</SUB>DP</UP> + E<UP> ∼ P      </UP>(<UP>reaction A</UP>)
          E<UP>∼P</UP>+<UP>N<SUB>2</SUB>DP ↔ N<SUB>2</SUB>TP</UP> + E<UP>      </UP>(<UP>reaction B</UP>)
<UP><SC>Scheme</SC> I</UP>
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 beta -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 Ndelta toward the phosphate, a well defined water molecule bridging it to the beta -phosphate oxygen in the ADP complex, at the presumed position of ATP gamma -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 alpha A/alpha 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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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 Delta 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 Delta 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 [gamma -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
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Abstract
Introduction
Procedures
Results
Discussion
References

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 (lambda 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 (lambda 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, open circle ; AZT-XP, black-triangle. Data were fitted with the following equation: Keq = ([E-P] [NDP])/([E] [NTP]) modified to the following formulation: Delta F = (Delta Fmax·r)/(r + Keq), where r = [NDP]/[NTP], Delta Fmax the maximum variation in fluorescence, and Delta 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.

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, open circle ; ddATP, triangle ; ddTTP, bullet ; ddCTP, black-triangle.

                              
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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.

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 (open circle ), ddTTP (bullet ), ddATP (triangle ), and ddCTP (black-triangle). 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 (open circle ), ddTTP (bullet ), ddATP (triangle ), ddCTP (triangle ), as well as AZTTP (black-down-triangle ). 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.

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 (bullet ) and AZTDP (black-down-triangle ).The apparent second order constant is 4500 M-1 × s-1 for ddTDP and 1500 M-1 × s-1 for AZTDP.

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 (bullet ) or ddADP (black-triangle) 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.

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.

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.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 beta  phosphate.

Our kinetic data are compatible with the following reaction.
E+<UP>ddNTP</UP> <LIM><OP><ARROW>↔</ARROW></OP><LL>k<SUB><UP>−1</UP></SUB></LL><UL>k<SUB><UP>+1</UP></SUB></UL></LIM> E · <UP>ddNTP</UP> <LIM><OP><ARROW>↔</ARROW></OP><LL>k<SUB><UP>−2</UP></SUB></LL><UL>k<SUB><UP>+2</UP></SUB></UL></LIM> E-<UP>P</UP> · <UP>ddNDP</UP> <LIM><OP><ARROW>↔</ARROW></OP><LL>k′<SUB><UP>+1</UP></SUB></LL><UL>k′<SUB><UP>−1</UP></SUB></UL></LIM> E-<UP>P</UP>+<UP>ddNDP</UP>
<UP><SC>Reaction</SC> 1</UP>
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. 
E+<UP>ddNTP</UP> <LIM><OP><ARROW>↔</ARROW></OP><LL>k<SUB><UP>−1</UP></SUB></LL><UL>k<SUB><UP>+</UP>1</SUB></UL></LIM> E · <UP>ddNTP</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB><UP>+2</UP></SUB></UL></LIM> E-<UP>P</UP>+<UP>ddNDP</UP>
<UP><SC>Reaction</SC> 2</UP>
When studying dephosphorylation by ddNDP in the absence of ddNTP, the same argument leads to writing the reaction mechanism as shown in Reaction 3. 
E-<UP>P</UP>+<UP>ddNDP</UP> <LIM><OP><ARROW>↔</ARROW></OP><LL>k′<SUB><UP>−1</UP></SUB></LL><UL>k′<SUB><UP>+1</UP></SUB></UL></LIM> E-<UP>P</UP> · <UP>ddNDP</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB><UP>−2</UP></SUB></UL></LIM> E+<UP>ddNTP</UP>
<UP><SC>Reaction</SC> 3</UP>
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).
k<SUB><UP>obs</UP></SUB>=<FR><NU>k<SUB><UP>+2</UP></SUB> · [<UP>ddNTP</UP>]</NU><DE>(k<SUB><UP>−1</UP></SUB>/k<SUB><UP>+1</UP></SUB>)+[<UP>ddNTP</UP>]</DE></FR> (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 approx  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 approx  2.5 s-1 and KS approx  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 approx 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 approx 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 approx  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 [gamma -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 beta - and gamma -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.

parallel 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
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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