From the Max-Planck Institut für biophysikalische Chemie, Abteilung Molekulare Genetik, D-37070 Göttingen, Germany and the § Max-Planck Institut für molekulare Physiologie, Postfach 102664, D-44026 Dortmund, Germany
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ABSTRACT |
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Nucleoside-diphosphate kinases (NDKs) catalyze
the transfer of -phosphoryl groups from NTPs via an active site
histidine to NDPs using a ping-pong mechanism. We have used the change
of intrinsic tryptophan fluorescence that occurs upon phosphorylation of NDK to measure the rates of phosphorylation and
dephosphorylation with a range of nucleotides and nucleotide
analogues. For natural nucleotides, the rates of phosphorylation
and dephosphorylation were linearly dependent upon nucleotide
concentration until they became too fast to measure. The second order
rate constants for phosphorylation by natural NTPs varied between 0.7 and 13 × 106 M
1
s
1. Dephosphorylation by NDPs was 2-3-fold faster than
the corresponding phosphorylation reaction, and dephosphorylation by
dNDPs was 3-4-fold slower than the equivalent NDPs. In all cases,
second order rate constants were highest for guanine followed by
adenine and lowest for cytosine nucleotides. NDK also catalyzes the
transfer of thiophosphate from adenosine
5'-O-(thiotriphosphate) (ATP
S) and guanosine
5'-O-(thiotriphosphate) (GTP
S) to NDP, but at
of the equivalent phosphoryl transfer rates. In this
case, the observed rate constants of phosphorylation and
dephosphorylation were hyperbolically dependent on nucleotide
concentration. Thiophosphorylation by ATP
S and GTP
S occurred with
kmax of 2.8 and 1.35 s
1 and
Kd of 145 and 36 µM respectively. For
dethiophosphorylation by a range of NDPs, kmax
was in the range of 5-30 s
1, whereas
Kd varied between 0.16 and 3.3 mM.
Guanine had the lowest Kd values, and cytosine had
the highest. The data are consistent with fast reversible binding of
the nucleotide followed by the rate-limiting phosphoryl transfer.
Thiophosphates change only the rate of the phosphoryl transfer step,
whereas both events are influenced by the base. Modification at the
2'-hydroxyl of ribose has only a small effect, while the overall rate
of phosphoryl transfer is reduced 1000-fold by modification at the
3'-ribose.
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INTRODUCTION |
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Nucleoside-diphosphate kinase
(NDK1; EC 2.7.4.6) is a
ubiquitous enzyme that catalyzes the transfer of the -phosphoryl
group from a nucleoside triphosphate to a nucleoside diphosphate (1), involving a phosphohistidine intermediate state (2). NDK exists as
either hexamers (most eukaryotic species) or tetramers (most prokaryotic species) and has been reported to display little
specificity for different nucleotide bases (2, 3), reflecting a pivotal role in maintaining balanced levels of all oxy- and deoxynucleoside triphosphates in the cell. It is thus considered a housekeeping enzyme
(4), a role that has taken on greater significance with the therapeutic
use of nucleotide analogues such as azidothymidine (AZT) as replication
inhibitors as these compounds must be phosphorylated in the cell before
becoming active (5).
In humans, two very closely (89% identity) related isoforms of NDK, designated NDK-A and NDK-B, were identified biochemically (6), which associate in vivo to form mixed hexameric isoenzymes. Evidence that NDK-A and -B may have a more complex role in the cell was provided when they were shown to be identical to the tumor suppressor proteins nm23-H1 and nm23-H2, respectively (7, 8). In addition, several point mutations in either isoform have been found in aggressive tumors (9-12), emphasizing the importance of the presence of functional NDK in the cell. Beyond their role as nucleotide kinases, a multitude of biological activities have been ascribed to these enzymes, ranging from their potential function as histidine protein kinases to the identification of NDK-B as a nucleic acid-binding protein acting as a transcription factor for the c-myc oncogene (for a review, see Refs. 13 and 14). Most recently, another member of the human nm23 tumor suppressor family, DR-nm23, has been identified; this protein seems to be involved in myeloid cell proliferation and differentiation (15). A fourth member, nm23-H4, might represent a mitochondrial isoform of human NDKs (16). Thus, the complete biological role of NDK is not clearly defined, a situation underlined by the poor understanding of the mechanism of substrate recognition and phosphate transfer by the protein.
The mechanism of the NDK activity has been extensively studied in the steady state, and a ping-pong bi-bi mechanism of NDK is well established (4, 17). Thus, the overall reaction is the sum of a donor and acceptor half-reaction, following the reaction scheme,
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We present here a detailed transient kinetic analysis of the human NDK-A and -B, which have been overexpressed in bacterial cells. Each half-reaction of NDK-catalyzed phosphoryl transfer was examined independently for a wide range of substrates and substrate analogues. The approach allowed the measurement of individual rate and equilibrium constants for the reaction shown above and demonstrated that for most nucleotides the enzyme binds the nucleotide rapidly and reversibly followed by rate-limiting phosphoryl transfer. In addition, the enzyme shows a significant preference for adenine and guanine nucleotides with the highest affinity for guanine nucleotides and fastest phosphoryl transfer for adenine nucleotides.
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MATERIALS AND METHODS |
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Chemicals--
Nucleotides, enzymes, and other reagents were
obtained from Sigma or Boehringer Mannheim, unless stated otherwise.
AZT-TP and 2'(3')-O-(N-methyl-anthraniloyl)-ATP
were generous gifts from Prof. R. S. Goody. When checked for
purity by HPLC analysis (see below), ATP, ADP, GTP, and GDP were found
to be more than 95% pure. ATPS and ADP
S were more than 90% pure
with the contaminating nucleotides ADP and AMP, respectively, at less
than 10%.
Proteins--
The cDNAs encoding NDK-A and -B (which were a
generous gift from Dr. M.-L. Lacombe) were cloned into the vector pJC20
(23) with and without an N-terminal histidine tag. The pJC20 HisN
vector contains the multiple cloning site of pET19b. It fuses the amino acid sequence MGH10ID4KHLD to the N terminus of
the protein. This sequence includes an enterokinase cleavage site. For
overproduction of recombinant proteins, these vectors were transformed
into BL21 (DE3) E. coli strain. A total of 5-10 1-liter
cultures were grown, and expression was induced with 1 mM
isopropyl -D-thiogalactoside for 5 h at 37 °C at
an optical density of 0.5-0.7 at 600 nm. Cells were harvested by
centrifugation for 15 min at 5000 rpm in a Sorvall RC-3B centrifuge and
broken down by freezing and sonication. The cell debris was pelleted by
centrifugation for 1 h at 11,000 rpm in an SS-34 rotor in a
Sorvall RC-5B. Unmodified proteins NDK-A and -B were purified and
characterized as described previously (24).
Steady-state Kinetics-- NDK activity was measured at 20 °C in a standard pyruvate kinase/lactate dehydrogenase-coupled enzyme assay (4) using 2 mM ATP and 0.5 mM dTDP as substrates in buffer A. The nucleoside diphosphate substrate for NDK was dTDP because this nucleotide is a poor substrate for pyruvate kinase. An activity of 1 unit is defined as the turnover of 1 µmol of substrate in 1 min per mg of protein. The coupled enzyme assay is not suitable for following the NDK reaction with ATP and GDP as substrates, since the latter is a good substrate for pyruvate kinase. This reaction was studied by HPLC. 1 ml of experimental buffer containing 0.5-5 nM NDK-B was incubated with 5 mM ATP and 1 mM GDP. Aliquots of 40 µl were removed at fixed time intervals, and the reaction was stopped by heating to 80 °C or by injecting directly into the HPLC column. Nucleotides were separated on a C-18 reversed phase column (250 × 4.6 mm, Bischoff Chromatography) filled with Hypersil 5.0 µm on a Beckman System Gold HPLC apparatus. The column was run at room temperature at an isocratic flow of 1.5 ml/min with 100 mM phosphate buffer, pH 6.5, 10 mM tetrabutylammonium bromide, 7.5% acetonitrile (modified after Ref. 28). Turnover rates were determined from the amount of generated GTP and ADP as a function of time.
Protein Fluorescence--
Steady-state measurements on intrinsic
tryptophan fluorescence were recorded on a Perkin-Elmer LS 3B
fluorimeter at 20 °C. The rates of nucleotide-induced protein
fluorescence changes were measured with a Hi-Tech Scientific SF-51 or
SF-61 stopped-flow rapid mixing device equipped with a 100-watt
xenon/mercury lamp and monochromator. Intrinsic tryptophan fluorescence
was excited at 295 nm and detected at 90° to the incident light after
passing through a WG 320 cut-off filter. A total of 512 12-bit data
points were collected using a DAS 50 analogue to digital converter in a
Hewlett Packard 486 computer running Hi-Tech software. 3-10 traces
were collected with the same solution, averaged, and fitted to a
single-exponential (Ft = Famplitude
ekobst + F
) function using a nonlinear least-squares
fitting routine. In all measurements, enzyme was mixed with at least
5-fold excess of nucleotides; if nucleotide concentrations over 1 mM were used, MgCl2 was added to ensure that
Mg2+ was present in excess. Concentrations always refer to
the concentrations of the reactants after mixing in the stopped-flow
spectrophotometer. Experiments were performed at 20 °C in buffer
A.
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RESULTS |
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Characterization of Expressed Proteins
Gel Electrophoresis-- Unmodified and N-terminally histidine-tagged NDK-A and -B were purified to greater than 90% homogeneity. The proteins (NDK-A, NDK-AHis, NDK-B, and NDK-BHis) migrated in SDS-polyacrylamide gel electrophoresis (15% SDS-polyacrylamide gel electrophoresis) as single bands at 20, 23, 19, and 22 kDa compared with calculated molecular weights (excluding the N-terminal methionine) of 17,017, 20,198, 17,167, and 20,348, respectively. Histidine-tagged proteins showed a faint second band running at the size of the untagged protein. This was probably due to cleavage of the tag at the enterokinase recognition site during the purification process; it contained less than 10% of the total protein.
Activity--
When tested for activity in the coupled enzyme assay
(with 2 mM ATP and 0.5 mM dTDP as substrates)
at 20 °C, all proteins had specific activities of 450 units, being
equivalent to turnover rates of 130 s1. Activities of
NDK-B and -BHis with 5 mM ATP and 1 mM GDP at 20 °C as determined by HPLC analysis were 120 and 136 s
1, respectively. This compares to values of 160 and 210 s
1 measured before (6) for NDK-A and -B with 1 mM ATP and 0.3 mM 8-bromo-IDP at 25 °C.
Steady-state Fluorescence Studies
When 50 µM ATP was added to 1 µM NDK-B, the intrinsic tryptophan fluorescence decreased by about 10%. When phosphorylated NDK-B was prepared, the addition of 50 µM ADP resulted in a 7% increase in fluorescence. The addition of ADP to NDK-B or the addition of ATP to phosphorylated NDK-B had no measurable effect on the fluorescence, apart from a small quench due to absorption of the nucleotide. This demonstrated that the observed fluorescence changes indicated the phosphorylation state of the enzyme and not nucleotide binding (22).
In a titration experiment, the tryptophan fluorescence (excitation, 295 nm; emission, 340 nm) of 11 µM NDK-B or
NDK-BHis was recorded at ATP concentrations between 0 and
300 µM (Fig. 1). The change
in fluorescence (F) was plotted against the added ATP
concentration and used to determine K50%, the
concentration of ATP at which half of the maximal fluorescence change
occurred, i.e. when half of the total protein is
phosphorylated. A correction was made for the linear decrease in
fluorescence due to absorption of the nucleotide by repeating the
titration with ADP. At [ATP]total = K50%, half of the NDK (initially 11 µM) is phosphorylated, and therefore [NDK] = [NDK~P] = [ADP] =
µM. These values were
used to calculate the overall equilibrium constant
Keq from the equation,
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(Eq. 1) |
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NDK-A showed a similar quenching in fluorescence upon adding ATP; however, the change in fluorescence was smaller (~7% upon addition of ATP) than for NDK-B. Due to this small signal, Keq was not determined for NDK-A.
Transient Kinetic Studies
Phosphoryl Transfer Reactions of NDK-A and -B with ADP and
ATP--
Since the addition of ATP leads to a 10% decrease in protein
fluorescence, this signal can be used to follow the rate of the ATP-induced phosphorylation reaction. Fig.
2a shows the changes in
fluorescence recorded upon mixing 0.5 µM
NDK-BHis with 10 µM ATP in the stopped-flow
fluorimeter. A 9% decrease in fluorescence was observed, which was
well described by a single exponential function with an observed rate
constant (kobs) of 88 s1. When ADP
replaced ATP, no significant change in fluorescence occurred. In an
analogous experiment, 0.5 µM phosphorylated
NDK-BHis (NDK-BHis~P) was mixed with 5 µM ADP and led to a 6% increase in fluorescence, with
kobs = 119 s
1 (Fig.
2b). The addition of ATP to phosphorylated enzyme produced no change in fluorescence.
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(Eq. 2) |
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Phosphoryl Transfer Reactions of NDK-B with Natural Substrates-- The reactions shown in Fig. 2, a and b, were repeated for a wide range of naturally occurring nucleotides and in each case, the reaction could be described by a single exponential. The plots of kobs against the nucleotide concentration are shown in Fig. 3. The apparent second order rate constants obtained from the slopes of the plots are summarized in Table II.
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(Eq. 3) |
Phosphorylation of NDK-B with Ribose-modified Nucleotides--
To
clarify the ability of NDK-B to recognize different substrates, we
examined the phosphorylation reaction for a series of NTPs modified on
the ribose ring. We compared ATP, 2'-deoxy-ATP (dATP), and 3'-deoxy-ATP
(Cordycepin). All nucleotides phosphorylated the protein as observed by
the increase in protein fluorescence, and kobs
was linearly dependent upon nucleotide concentration. The apparent
second order rate constants were 7.9 × 106, 1.1 × 106 and 2 × 103
M1 s
1, respectively. In
contrast, the addition of 1 mM 2',3'-dideoxy ATP gave no
increase in protein fluorescence, indicating that it may not be a
substrate for NDK. The reaction of 3'-azido-3'-deoxythymidine triphosphate (a substrate for human immunodeficiency virus reverse transcriptase, leading to chain termination) with NDK gave an apparent
second order rate constant of 7 × 102
M
1 s
1, which is
of
the rate with 3'-dATP, showing that the bulky azide group can be
accommodated comparably well into the binding cleft. A similar result
was observed for methyl anthraniloyl ATP (mixed 2'- and 3'-isomers),
which phosphorylated NDK at
of the rate of 2'-dATP. These
results are in broad agreement with those of Bourdais et al.
(32) using a steady-state approach.
Phosphoryl Transfer Reaction with Thiomodified Nucleotide
Analogues--
Thiomodified nucleotides (ATPS and GTP
S) are
substrates for NDK (33), and indeed the thiophosphorylated enzyme can
be isolated and is as stable as the natural complex. This modification (substitution of sulfur for oxygen) has been shown to reduce the rate
of phosphoryl transfer for a wide variety of enzymes (34).
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(Eq. 4) |
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DISCUSSION |
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Human NDK-A and -B have been expressed and purified from Escherichia coli as both unmodified and His-tagged proteins. All four constructs behave in a similar way, suggesting that the His tag has no major influence on the activity of the proteins in the steady state. Since the NDK-BHis was the easiest to purify, the detailed kinetic analyses were all performed on this construct.
Phosphoryl Transfer Measured by the Intrinsic Fluorescence Change-- We have shown that, as for the D. discoideum protein (22), phosphorylation of the active site histidine in the human enzyme (His118) results in a quenching of the intrinsic protein fluorescence, which is reversed on dephosphorylation. Human NDK-A and -B contain three tryptophan residues at positions 78, 133, and 149 (35). The D. discoideum NDK has only a single tryptophan at position 137 (equivalent to position 133 in human NDK) and shows a larger (20%) quench of fluorescence on phosphorylation (22). This suggests that the 10% fluorescence quench of the human enzyme originates primarily from the Trp133. Since this Trp is conserved in almost all eukaryotic species, a similar fluorescence change may expected for other NDKs, so the transient kinetic approach developed here may be widely applicable.
We used this fluorescence change to study the mechanism of the enzyme with transient kinetic methods. Unlike steady-state methods, this approach can study each half-reaction independent of the competing back reaction. This is a major advantage for an enzyme with a ping-pong mechanism. All of the kinetic data shown here are consistent with reaction Scheme 1. The fluorescent transients observed for all four NDK constructs in the stopped-flow studies were single exponentials, and kobs was linearly dependent on nucleotide concentration over the accessible range (between 2 and 10 µM for GTP). Thus, the observed event is limited by the access of the nucleotide to the protein. However, except for ADP and GDP, each of the second order rate constants was significantly lower than that expected for a diffusion-controlled reaction. This point is emphasized by the results with other natural (Table II) and nonnatural (Table III) nucleotides in which the constant varied by more than a factor of 100 and yet all nucleotides had a similar diffusion coefficient. Thus, each half-reaction is most probably a two-step process: initial binding of nucleotide followed by phosphoryl transfer (or a conformational change of the protein, which limits phosphoryl transfer rate) as shown in Scheme 1. If nucleotide binding is much faster than the phosphoryl transfer step, then the second order rate constants in Tables I and II are defined by K1k+2 for phosphorylation and kNucleotide Binding and Dissociation Compared with the Phosphoryl
Transfer Step--
The presence of the thiophosphate has a marked
effect on phosphoryl transfer steps (33), so the 500-1000-fold lower
values of second order rate constants observed for the thiophosphate compared with the equivalent phosphate primarily reflect a change in
k+2 or k2 with a
relatively small change in the values of K0.5
(and hence K1 and K3).
This interpretation is supported by the data for dephosphorylation of
NDK by ADP
S and GDP
S, where K0.5 values
are similar to those for dethiophosphorylation by ADP and GDP. With the
assumption that K1 and K3
are similar for phosphoryl and thiophosphoryl transfer reactions, the
values of k+2 (or k
2)
can be predicted. These are 1200, 16,500, 460, and 4800 s
1 for ATP, ADP, GTP, and GDP, respectively. Similar
calculations can be made for all of the other nucleotides in Tables II
and III and suggest that for UDP, CDP, dADP, and dTDP
k
2 is similar to that of GDP (3000-6000
s
1) and is 500-1000 s
1 for dGDP, dUDP, and
dCDP. If we assume that nucleotide binding is diffusion-controlled
(k+1 = k
3 = 107 to 108 M
1
s
1), then it is possible to estimate the nucleotide
dissociation rate constants (k
1 = k+1/K1,
k+3 = K3k
3). The values of
1/K1 and K3 (equal to
K0.5) fall into two ranges: 50-150
µM (GTP, ATP, GDP, and dGDP) and >1 mM
(other nucleotides). Thus, k
1 for ATP and GTP
(and k+3 for GDP and dGDP) are expected to be in
the range 103 to 104 s
1 and
104 to 105 s
1 for all other NDPs.
Thus, the assumption of rapid equilibrium binding followed by
rate-limiting phosphoryl transfer will be valid for all dNDPs, UDP, and
CDP. In the other cases, the rate constant of nucleotide dissociation
is 1-10 times the rate constant of the following phosphate transfer
step; i.e. if the diffusion-controlled rate constant is
108 M
1 s
1, the
assumption of rapid equilibrium binding is valid for all nucleotides.
The above interpretation suggests that the phosphoryl transfer step
will be too fast to measure by stopped-flow methods for the reactions
with all NDPs. For NTPs, apart from GTP, the affinity may be too weak
to allow the reaction to be saturated at a reasonable nucleotide
concentration. The prediction that the fluorescence transient for GTP
should saturate at a kmax of 460 s
1 led us to examine this reaction more carefully.
Whereas all other kobs values were linear over
the accessible range of nucleotide concentrations, those for GTP showed
some evidence of saturation of kobs at above 20 µM. The reaction is difficult to measure because of the
small amplitude, but we were able to collect data up to 80 µM. The data points had large errors but showed a clear
deviation from a linear fit. A hyperbolic fit to the data suggested a
K0.5 of 15-35 µM and a
kmax of 250-500 s
1. While the
data are not conclusive, they are compatible with the prediction from
the GTP
S data.
Influence of Base and Sugar Moieties on Nucleotide Binding and Phosphoryl Transfer-- The results for the wide range of nucleotides (Fig. 3, Tables II and III) show clear differences in the way the base is recognized by NDK-B. The apparent second order rate constants were always highest with guanine nucleotides, followed by adenine and uracil nucleotides, and slowest with cytosine nucleotides with the relative rates of 1, 0.55, 0.12, and 0.04. Furthermore, dephosphorylation was always twice as fast as phosphorylation, with the same nucleotide giving the expected invariant Keq. Thus, although the free energy of phosphoryl transfer is the same in each case, the enzyme clearly distinguishes the different bases. The discrimination between the bases by NDK has not been well established before, because the problem with competitive inhibition between substrates mentioned in the introduction and the coupled enzyme assay, which is predominantly used to measure steady-state activity, allows only certain pairs of substrates to be used. Thus, it was often claimed that NDK is nearly completely unspecific (3, 36, 37), despite some studies showing highly varying values of Km with different nucleotides for human NDK (18).
Studies with thiophosphoryl nucleotides reveal that the effects of the base on the second order rate constants contain a contribution from both a change in the affinity of the nucleotide and a change in the phosphoryl transfer step. Thus, it was observed that at low nucleotide concentrations, GTP and GDP were the preferred substrates because of high affinity (low K0.5), but ATP and ADP have the highest kmax. The data in Table III show that changes in the base can have marked effects on both K0.5 and kmax but that effects on K0.5 are greater (e.g. 20-fold compared with 6-fold for NDPs). Similarly the loss of the 2'-hydroxyl on the ribose causes an 8-fold reduction in kmax and in each case a small increase in K0.5 for dADP, dGDP, and dCDP but a significant decrease for dUDP.Structure-Function Relationships--
The interactions of the base
and the sugar of the nucleotide with NDK have been revealed in crystal
structures of NDK with bound dTDP (NDK of D. discoideum
(37)), ADP (NDK of D. discoideum (36); NDK of
Myxococcus xanthus (38)), and GDP (NDK-B (39)). The base of
the nucleotide is wedged between Phe60 and
Val112, and Glu152 (numbering refers to human
NDK) can form a hydrogen bond directly with GDP or via a water molecule
with ADP and dTDP. This interaction may help to discriminate guanine
from other bases (39) and lead to lower values of
K0.5 reported here for guanine nucleotides. The
ribose 2'- and 3'-hydroxyls of ADP and GDP form hydrogen bonds with
Lys12 and Asn115 (39). With dTDP, the
interactions of the 3'-hydroxyl were conserved, and a water molecule
replaced the hydrogen bond of 2'-hydroxyl to Lys-12 (37). The loss of
the 3'-hydroxyl has a much more severe effect on the second order rate
constant as shown in this study and in steady-state assays (32).
Bourdais et al. (32) propose that the poor activity with
3'-modified nucleotides is not due to a lack of binding but rather to
an internal bond of the 3'-OH with the -phosphate, which is needed
to place the
-phosphate correctly to be attacked by the N-
nitrogen of the catalytic histidine.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. Reinstein and Prof. I. Lascu for helpful comments on the manuscript. We are grateful to Profs. D. Gallwitz and R. S. Goody for continuous support.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft and the Max-Planck Society.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. E-mail:
mkonrad{at}gwdg.de.
1
The abbreviations used are: NDK,
nucleoside-diphosphate kinase; ATPS, adenosine
5'-O-(thiotriphosphate); GTP
S, guanosine 5'-O-(thiotriphosphate); NTP
S, nucleoside
5'-O-(3-thiotriphosphate); GDP
S, guanosine
5'-O-2-(thio)diphosphate; ADP
S, adenosine
5'-O-2-(thio)diphosphate; NDP
S, nucleoside
5'-O-2-(thio)diphosphate; AZT-TP, 3'-Azido-3'-deoxythymidine triphosphate; AZT-DP, 3'-Azido-3'-deoxythymidine diphosphate; HPLC,
high pressure liquid chromatography; MOPS, 4-morpholinepropanesulfonic acid; NDK-BHis~thioP, thiophosphorylated
NDK-BHis.
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REFERENCES |
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