(Received for publication, January 16, 1996; and in revised form, February 5, 1996)
From the
Nucleotide analogs are widely used in antiviral therapy and particularly against AIDS. Delivered to the cell as nucleosides, they are phosphorylated into their active triphospho derivative form by cellular kinases from the host. The last step in this series of phosphorylations is performed by nucleoside diphosphate (NDP) kinase, an enzyme that can use both purine or pyrimidine and oxy- or deoxynucleotides as substrates.
Using pure recombinant human NDP kinase type B (product of the gene nm23-H2), we have characterized the kinetic parameters of several nucleotide analogs for this enzyme. Contrary to what is generally assumed, diphospho- and triphospho- derivatives of azidothymidine as well as of dideoxyadenosine and dideoxythymidine are very poor substrates for NDP kinase. The rate of phosphorylation of these analogs varies between 0.05% and 0.5%, as compared to the corresponding natural nucleotide, a result that is not due to the inability of the analogs to bind to the enzyme. Using the data from the high resolution crystal structure of NDP kinase, we provide an interpretation of these results based on the crucial role played by the 3`-OH moiety of the nucleotide in catalysis.
The replicative cycle of human immunodeficiency viruses (HIV), ()involves the action of a reverse transcriptase that is an
important target for chemotherapeutic intervention. Indeed, nucleoside
analogs substituted on the 3`-OH of the ribose such as azidothymidine
(AZT) and dideoxyinosine (ddI) are powerful inhibitors of this enzyme;
they act as chain terminators inhibiting virus replication
specifically, due to the fact that cellular DNA polymerase binds to
these analogs with a low affinity as compared to viral reverse
transcriptase(1, 2) . However, to be active,
nucleoside analogs need to be phosphorylated into triphosphonucleotides
by cellular kinases since HIV does not carry genes for enzymes that
metabolize purine and pyrimidine nucleotides. The reactions leading to
mono- and diphosphates of the nucleosides are catalyzed by
base-specific enzymes, i.e. the phosphorylation of purines and
pyrimidines in the cell is catalyzed by distinct nucleoside kinases and
nucleoside monophosphate kinase. In contrast, the step leading from the
nucleoside diphosphate to the triphosphate is catalyzed by a single
enzyme, nucleoside diphosphate (NDP) kinase, independent of the nature
of the base and of the sugar (EC 2.7.4.6)(3) .
The main function of NDP kinase in the cell is to phosphorylate the non-adenine nucleoside diphosphates into triphosphates. The reaction has a ping-pong mechanism, with a phosphohistidine intermediate according to the following reactions ( and ).
ATP is believed to be the main phosphate donor in the cell. Renewed interest in this enzyme resulted recently from its cloning from several species including the prokaryote Myxococcus xanthus(4) , the primitive eukaryote Dictyostelium discoideum(5) , and higher eukaryotes including mammals. Two highly homologous NDP kinases, NDPK-A and NDPK-B, have been isolated in human erythrocytes and sequenced(6) , and these proteins were identified to the products of the genes nm23-H1 and nm23-H2, respectively (6, 7) . nm23-H1 has been shown to be involved in tumor metastasis(8, 9) . All NDP kinases are made of identical 17-kDa subunits. Eukaryotic NDP kinases are hexamers, whereas some bacterial enzymes are tetramers. The high resolution structure of the NDP kinases from Dictyostelium(10, 11) , M. xanthus(12) , Drosophila(13) , and human (14, 15) show that the subunit fold and active site of NDP kinases are highly conserved throughout evolution. This fold is original for a phosphotransferase, showing no similarities with the usual nucleotide binding fold of nucleotide-binding proteins. High resolution data are also available for Dictyostelium and Myxococcus NDP kinase complexed with ADP, a purine nucleotide(15, 16) , for Dictyostelium complexed with TDP, a pyrimidine deoxynucleotide(17) , and for human NDPK-B complexed with GDP(14) . These data, along with the study of several mutant proteins modified in active site residues by in vitro mutagenesis(18) , provide a comprehensive description of the active site.
Nucleoside analogs are thought to be phosphorylated by the same enzymes as the natural nucleotides. For example, thymidine kinase and thymidylate kinase catalyze the first and second steps in the phosphorylation of AZT. However, AZT-MP is a poor substrate for thymidylate kinase and accumulates in the cell(19) , which may be responsible for a major part of its cytotoxic effects(20) . In contrast to the numerous studies performed on AZT phosphorylation to AZT-MP and AZT-DP by thymidine kinase and thymidylate kinase, no study is available on the last step in the phosphorylation cascade, i.e. the phosphorylation of AZT-DP in AZT-TP. This may be due to the lack of specificity of NDP kinase toward the nucleobase of natural nucleotides, which has led to the general assumption that this enzyme would also easily phosphorylate diphosphates of nucleoside analogs and in particular AZT-DP and ddADP. However, the cellular concentration of AZT-TP is even lower than that of AZT-DP, unlike ATP which is much more abundant than ADP(19) . This suggested to us that AZT-DP may be a poor substrate for NDP kinase and that the reaction catalyzed may be a second limiting step in the phophorylation pathway.
In this paper we have investigated the ability of antiviral diphospho- and triphosphonucleotides to be used as substrates by human NDP kinase. The results are discussed in the context of the crystal structure of NDP kinase and in particular of the role played by the 3`-OH of the ribose moiety in substrate binding and in catalysis.
To synthesize phosphoderivatives of AZT, the free 5`-OH of AZT was
phosphorylated by condensation with -cyanoethyl dihydrogen
phosphate (22) in the presence of DCC in anhydrous pyridine to
give the phosphodiester, followed by treatment with 0.4 LiOH for 1 h.
AZT-DP and AZT-TP were obtained one-pot from AZT-MP via the
phosphoroimidazolate prepared from the phosphomonoester and
1,1`-carbonyldiimidazole(23) . The di- and triphosphate were
isolated by chromatography on a DEAE-Sephadex A-25 column
(HCO
form) eluted with a linear gradient
of triethylamonium hydrogen carbonate buffer (pH 7-8;
0.05-0.5 M).
ddADP was enzymatically synthesized from
ddATP in presence of 3-fold excess fructose 6-phosphate and
phosphofructokinase in 50 mM Tris-HCl (pH 8), 5 mM MgCl for 3 h at 20 °C. It was purified by
reversed-phase chromatography on a C-18 column eluted with
acetonitrile-water (0-25%).
AZT-DP, AZT-TP, and ddADP were
repurified by reversed-phase high performance liquid chromatography
(Nucleosil 100, 5 µm, 250 mm 10 mm; A = 0.01 M TEAA, B = MeCN from 0-20% in 20 min, flow rate 5
ml/min), and their purity was checked by
H,
C,
and
P NMR and by mass spectrometry (fast atom
bombardment).
The assays were started by adding 3 µl of
enzyme to a reaction mixture (10 µl) containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl, and the
substrates at 37 °C. The amount of NDP kinase added per assay
varied from 10 pg for natural nucleotides to 5 ng with poor analogs.
When the nucleotide triphosphates were assayed, the reaction was
stopped by adding 3-µl aliquots to a 2-µl stop solution
consisting of 0.7 M formic acid and 10 mM each of ADP
and ATP. When assaying nucleotides diphosphates with
[
-
P]GTP, the reaction was stopped by
placing a 3-µl aliquot of the reaction mixture at 85 °C for 2
min. After cooling on ice, 2 µl of a 10 mM solution of
cold nucleotide was added. The nucleotides were separated on TLC plates
with UV indicator (Macherey-Nagel, Germany) which were developed with
400 mM NH
HCO
or 1 M formic
acid and 1.5 M LiCl when [
-
P]GTP
or [
C]ATP were used, respectively. The products
formed were quantified with the WIN-IQ program (Molecular Dynamics)
using a PhosphorImager screen. Linear readings of the radioactivity
were obtained in a range covering 5 orders of magnitude in nucleotide
concentration. Kinetic parameters were calculated by nonlinear fitting
using Kaleidagraph software.
We have investigated the ability of NDP kinase to use the diphosphate and triphosphate forms of AZT, ddA and ddT, as phosphate acceptor and phosphate donor, respectively. Since the two human isozymes of NDP kinase, NDPK-A and NDPK-B, do not differ in their enzymatic properties(6) , we have used only the isozyme NDPK-B encoded by the gene Nm23-H2(9) , to perform the experiments reported in this paper. Preliminary experiments using NDPK-A gave similar results (data not shown).
Fig. 1shows a
typical kinetic experiment. The rate of product accumulation was
constant for at least 6 min, allowing determination of initial
velocities (Fig. 1, inset). It should be noted that the K and V
values derived from
these experiments are apparent kinetic parameters measured by varying
the concentration of one substrate only. Due to competition between the
nucleoside di- and triphosphates, inhibition by excess of substrate
makes a more complete study difficult. However, for an enzyme with a
ping-pong mechanism, the ratio of the apparent k
/K
is equal to the true
value of k
/K
; therefore, it
is a useful parameter when comparing the natural substrates to the
analogs.
Figure 1:
Measure of kinetic parameters. Initial
velocities were determined in the presence of 0.1 mM ADP from
the slope of the curves shown in inset. The apparent V for ATP and the apparent K
were computed from this plot using the software
Kaleidagraph. Inset, time course of
[
C]ATP formation from
[
C]ADP (0.1 mM) in the presence of 0.25
mM (
), 0.5 mM (
), 1 mM (
), or 2.5 mM (
)
ATP.
As shown in Table 1and Table 2, AZT
nucleotides are very poor substrates for the NDP kinase reaction. When
used in the diphospho- form as an acceptor, the apparent k is 0.17% of that of TDP (Table 1), while
it is 0.05% of that of TTP when used in the triphospho- form as the
phosphate donor (Table 2). The ratio k
/K
is high with natural
nucleotides, actually close to the value predicted for
diffusion-controlled reactions. It drops by several orders of magnitude
for all analogs with a modified 3`-OH position on the ribose moiety.
This is true, for instance, for analogs in which the 3`-OH is missing,
such as 3`-dATP (which yields only 0.4% of the activity with ATP) or
the dideoxy analogs ( Table 1and Table 2). Very low k
are measured when ddTTP or ddATP is used as
donor (0.01% and 0.04% of TTP and ATP, respectively), or when ddADP is
used as the acceptor (0.4% of ADP). These results point to the
importance of the 3`-OH group as opposed to the 2`-OH. It is
interesting to note that similar results were obtained with 3`-dATP and
AZT-TP, suggesting that steric hindrance by the bulky azido group in
AZT nucleotides is not the reason for their poor performance as
substrates of NDP kinase. In contrast, preliminary measurements showed
that arabino-ATP (where the sugar moiety is the epimer of ribose in the
2` position) is a good substrate for NDP kinase (data not shown).
We
also performed experiments where the analogs were tested as competitors
in the reaction of phosphorylation of [C]ADP by
TTP. AZT-TP and ddTTP were both inhibitors (data not shown), with
I
values approximately equal to their apparent K
(see Table 1and Table 2),
indicating that a lack of binding to the enzyme is not the reason of
the poor activity described above. Under the conditions used, no
transfer of
-phosphate from either analog to ADP could be
detected.
The x-ray structures of several NDP kinases in complex
with nucleotides explain the lack of specificity of the enzyme for the
nucleo-base. Unlike most nucleotide-binding proteins, NDP kinase does
not form specific hydrogen bonds with the base (Fig. 2). In
contrast, there is extensive bonding to the 3`-OH of the sugar, which
accepts hydrogen bonds from the Lys-16 and Asn-119 side chains (numbers
correspond to the Dictyostelium NDP kinase sequence). The role
of these amino acids has been confirmed by site-directed
mutagenesis(18) . Moreover, the 3`-OH donates a hydrogen bond
to one of the -phosphate oxygens(16, 17) . This
internal bond maintains the nucleotide in a folded conformation, which
is probably needed to position the
-phosphate correctly for
in-line attack by the N
nitrogen of the catalytic histidine. Its
presence also suggests that the 3`-OH plays a role in catalysis by
donating its proton to the leaving group and helping release of the
nucleoside diphosphate product. Our data on the study of nucleoside
analogs support this suggestion.
Figure 2:
The
nucleotide binding site in NDP kinase. Stereoview of the active site in
a subunit of Dictyostelium NDP kinase with bound
TDP(17) . His-122 interacts with Glu-133 on top. The thymine
base on bottom is caught in a slit between the Phe-64 and Val-116 side
chains. It points toward outside the protein and makes no polar
interaction with it. The 3`-OH group on the deoxyribose receives
hydrogen bonds from Lys-16 and Asn-119 and donates one to the
-phosphate. The latter also interacts with Arg-92 and Arg-102. A
Mg
ion bridges the
- and
-phosphates. The
active site structure is essentially unchanged in the phosphorylated
enzyme (25) and in human
NDPK-B(14) .
We have shown that the di- and triphosphate forms of AZT, ddA
and ddT, are poor substrates for NDP kinase and that the absence of a
3`-OH on the sugar is largely responsible for their lack of activity.
These results are in agreement with previous studies showing some in vivo accumulation of the AZT-DP (19) and
dideoxynucleotides in MT-4 cells(24) . Although they suggest
the possibility that these and other nucleoside analogs lacking a 3` OH
group such the acyclic nucleosides, may not be phosphorylated by NDP
kinase in vivo, it should be kept in mind that the turn over
of NDP kinases is unusually high (more than 1000 s),
and therefore that even poor substrates may be phosphorylated in the
cell. Our results may help understanding the pharmacokinetics of
nucleoside analogs. They may provide a rational basis for the drug
design of new active molecules, with the hope that analogs more
efficiently phosphorylated by NDP kinase can be used at a lower dose
and elicit less toxic and secondary effects.