Background
Ecto-Nucleoside triphosphate diphosphohydrolases
(E-NTPDases, formerly called ecto-ATPases) hydrolyze
nucleotides in the presence of divalent cations and are
insensitive to inhibitors of P-type, F-type, and V-type
ATPases [ 1 ] . Three isoforms that differ in the ratio of
ATPase/ADPase activity are present on the cell surface [ 2
] : E-NTPDase1 with a ratio of 1, E-NTPDase2 with a ratio
of 10 and E-NTPDase3 with a ratio of 3-5. NTPDases are
important in many physiological processes like cell
motility, adhesion, nonsynaptic information transfer,
secretion, regulation of hemostasis and ectokinases [ 1 ] .
Understanding the enzymatic mechanisms of the NTPDases will
help description of their physiological functions, and
development of strategies to regulate the functions of the
enzymes.
The catalytic mechanism of NTPDases is not known even
though some basic facts of the catalysis have been
established. NTPDases do not form phosphorylated
intermediates during catalysis, a conclusion also supported
by lack of vanadate sensitivity and Pi product inhibition [
3 4 5 6 ] . The catalytic reaction appears to be
irreversible and no partial reactions have been observed [
7 8 ] . Divalent cations like Ca 2+or Mg 2+are required for
activity, and maximal activity is reached when the
concentrations of substrates and divalent cations are equal
[ 1 ] . The specific activities of NTPDases vary over a
broad range from ten thousand units for potato apyrase to
less than one hundred units for chicken gizzard ecto-ATPase
[ 9 10 ] . Sequence comparisons indicate that most of
NTPDases contain five highly conserved regions, apyrase
conserved region, ACR1 - ACR5 [ 9 11 ] . However, the
catalytic sites have not been identified, although ACR1 and
ACR4 have been implicated in β- and γ-phosphate binding,
respectively [ 9 ] .
E-NTPDase1 is also called CD39, as it was first
described as an antigen present on activated B and T
lymphocytes. Residues of ACR1 to ACR5 of CD39 have been
mutated to study the involvement of the ACR regions in
catalysis. E174 in ACR3 and S218 in ACR4 are required for
catalytic function [ 12 ] . Substitution of H59 in ACR1
converted CD39 into an ADPase in a quaternary structure
dependent manner [ 13 ] . Mutation of W187A in ACR3
affected CD39 folding and translocation, while mutation of
W459A in ACR5 increased ATPase activity but diminished
ADPase activity [ 14 ] . Mutations of D62 and G64 of ACR1
and D219 and G221 of ACR4 demonstrated that the nucleotide
phosphate binding domains of NTPDases are similar to those
present in the actin/heat shock protein/sugar kinase
superfamily [ 15 ] . These results suggest that the
conserved residues of the ACR1 to 5 regions are involved in
the catalytic mechanism of CD39.
The catalytic activity of CD39 is dependent on the
presence of divalent cations. Since the interactions of Ca
+2and Mg +2with proteins are difficult to study due to the
lack of spectroscopic properties, vanadyl (V IV=O) 2+has
been used as a probe of the ligands that compose Mg 2+, Ca
2+, and Mn 2+binding sites of several proteins, including
carboxypeptidase [ 16 ] , S-adenosylmethionine synthetase [
17 18 ] , pyruvate kinase [ 19 20 ] , and F
1 -ATPase [ 21 22 ] . This cation
specifically binds to divalent cation binding sites of
several enzymes, and in many cases serves as a functional
cofactor [ 23 ] . Vanadyl has one axial and four equatorial
coordination sites relative to the axis of the
double-bounded oxygen, an arrangement that is similar to
that for Ca 2+and Mg 2+. As it is known that the
A and
g tensors derived from the EPR spectrum
of bound VO 2+are a direct measure of the nature of the
equatorial metal ligands [ 24 ] , binding of VO 2+to CD39
could provide details about the catalytic mechanism of
CD39.
Recently we reported that a recombinant soluble CD39,
capable of hydrolyzing both ATP and ADP, was expressed and
purified from insect cells [ 25 ] . Only one
nucleotide-binding site was identified on the purified
soluble CD39 in the presence of Ca 2+when non-hydrolysable
nucleotide analogs were used. In this report, we
characterized the signals that were obtained from bound VO
2+when ATP or ADP was present at the catalytic site of the
purified soluble CD39. The possible metal ligands for VO
+2at the catalytic site are proposed and the catalytic
mechanism is discussed.
Results
Nucleotidase activity of purified soluble CD39 with
VO 2+as cofactor
The ability of purified soluble CD39 to hydrolyze VO
2+ATP is shown in Figure 1. Soluble CD39 did not
hydrolyze either ATP or ADP in the absence of VO 2+(Fig.
1A). When VO 2+was mixed with ATP at a ratio of 1:1, the
concentrations of both ADP and AMP increased and ATP
decreased as the incubation time was prolonged (Fig. 1B).
The ATPase activity of sCD39 with VO 2+was about 25% of
that with Ca 2+as a cofactor. Vanadyl is unstable in
aqueous solution at pH7.0 in the absence of chelator and
will precipitate out of solution as [VO(OH)
2 ]
n . The rate of precipitation depends
on the abundance and affinity of the chelator. This means
that the actual VO 2+concentration was lower than 0.5 mM.
This result indicates that VO 2+can functionally
substitute for Ca 2+as cofactor for sCD39 nucleotidase
activity.
Characterization of bound VO 2+ADPNP by
CW-EPR
The parallel features of CW-EPR spectrum of bound VO
2+in the presence of ADPNP, an ATP analog, are shown in
Figure 2a. This spectrum shows 51V hyperfine splitting
and the center of the parallel transitions from molecules
with the V=O bond oriented along the magnetic field (A
|| , g
|| ) which are strong enough to tell
the nature of VO 2+equatorial ligands [ 22 ] . Of the
eight transitions that result from the parallel oriented
molecules, the -7/2
|| , -5/2
|| , +3/2
|| , +5/2
|| , and +7/2
|| transitions (shown in the figures
from left to right, respectively) do not overlap with
perpendicular transitions. The 51V hyperfine splitting
spectra from molecules with V=O bond perpendicular to the
magnetic field (A⊥) are much smaller and not shown here [
21 ] . The intensity of -5/2
|| peak is used as direct measurement
of the amount of bound VO 2+, since this peak is the most
intense peak in the EPR spectrum that contains
contribution only from A
|| but not A⊥ [ 21 26 ] . In this
study, the intensities of each bound VO 2+-EPR feature
were normalized to 1 mg of protein.
VO 2+bound as the VO 2+-AMPPNP complex to sCD39
produced a strong spectrum characterized by A
|| of 504.25 MHz and g
|| of 1.9410 (Fig. 2b), called species
T (Table 1). The best fit of EPR species T to eq 1 is one
equatorial nitrogen from an amino group and three
equatorial oxygen ligands from carboxyl or phosphate
groups (Table 2). This result is consistent with AMPPNP
binding strongly to a single site on sCD39 in the
presence of metal [ 25 ] .
Characterization of EPR species from VO 2+-AMPCP
bound to sCD39
Figure 3ashows the parallel features of the EPR
spectrum of sCD39 bound VO 2+-AMPCP. Two sets of parallel
transitions were observed, and the derived A
|| and g
|| values are listed in Table 1. One
set had A
|| of 521.78 MHz and g
|| of 1.937, which is defined as
species D1 (Fig. 3b). The other set displayed A
|| of 490.01 MHz and g
|| of 1.9435, which is called species
D2 (Fig. 3c). The intensity of species D1 accounted for
11.4% of species T from bound VO 2+-AMPPNP, and the
intensity of D2 accounted for 7.1% of species T. The
intensity ratio of species D1 over D2 was 1.6.
In order to distinguish species D1 from D2, the sample
with VO 2+-AMPCP bound to sCD39 was thawed and incubated
at room temperature for 30 minutes, and the VO 2+EPR
spectrum was collected again. As shown in Figure 4aand
4b, either A
|| or g
|| values for both species D1 and D2
were changed. The intensity of species D1 was not changed
as it accounted for 12.1% of the intensity of species T
of the bound VO 2+-AMPPNP. However, the intensity of
species D2 was decreased dramatically, and it accounted
for only 0.1% of the intensity of species T. The
intensity ratio of D1 over D2 increased about 75 fold to
become 120.
There are two sets of equatorial ligands that can fit
well the EPR species D1 according to Eq 1 (Table 2). One
set includes two equatorial oxygen from two water
molecules, one equatorial oxygen from a carboxyl group or
phosphate, and one equatorial nitrogen from an amino
group. The other set contains one equatorial oxygen from
water and three equatorial oxygens from carboxyl groups
or phosphate. The best fit for the EPR species D2 to eq 1
is one equatorial oxygen from a hydroxyl group and three
equatorial oxygens from carboxyl groups or phosphate.
EPR characteristics of sCD39 bound VO 2+-ATP
In order to capture the bound VO 2+-EPR signal before
the enzyme completely turned over, sCD39 and VO 2+-ATP
were mixed on ice, immediately transferred into the EPR
tube and frozen. The entire process took about 15
seconds. The parallel portion of the collected VO 2+-EPR
spectrum is shown in Figure 5a. VO 2+-ATP complex bound
to sCD39 produced an EPR spectrum with A
|| of 489.5 MHz and g
|| of 1.9455, which corresponded to
species D2 (Fig. 5b). The signal intensity from the bound
VO 2+-nucleotide complex accounted only for 7.5% of that
of species T from bound non-hydrolysable VO 2+-ADPNP
complex.
The same sample made from mixing VO 2+-ATP and sCD39
was incubated at room temperature for 30 minutes, then
the VO 2+-EPR spectrum was generated as shown in Figures
4cand 4d. The EPR parameters derived from this VO 2+-EPR
spectrum were 489.5 MHz for A
|| and 1.9455 for g
|| respectively, which is consistent
with species D2. However, the signal intensity decreased
about 37.5 fold compared to that obtained before room
temperature incubation.
Free VO 2+binding to sCD39 characterized by
CD-EPR
Like other metals (Ca 2+and Mg 2+), free VO
2+inhibited the nucleotidase activities of sCD39 at high
concentration (data not shown). VO 2+in the absence of
any nucleotides was added to sCD39 at 1:1 molar ratio.
The parallel transitions of bound VO 2+-EPR spectrum are
shown in Figure 6. The features derived from the VO
2+-EPR spectrum were 486 MHz for A
|| and 1.946 for g
|| , which was designed as species V
(Fig. 6b). The signal intensity of bound VO 2+accounted
for 20.3% of that from the bound VO 2+-ADPNP complex.
The best fit of equatorial ligands for species V
according to eq 1 is two equatorial oxygen from hydroxyl
groups and another two equatorial oxygen from two water
molecules.
Discussion
Vanadyl has been used to estimate the types of groups
that serve as metal-ligands in F
1 -ATPase and other enzymes [ 16 18 19
21 ] because the g and A tensors of the 51V hyperfine
couplings are approximately a linear combination of tensors
from each type of group that contributes an equatorial
ligand [ 24 27 ] . By studying the EPR spectra of bound VO
2+in the presence of different nucleotides, we show that
the interaction of soluble CD39 with ATP is different from
that with ADP.
It is not surprising that VO 2+can functionally replace
Ca 2+in the hydrolysis of both ATP and ADP by soluble CD39,
although the enzymatic activity is about 25% of that with
Ca 2+as the cofactor, since F
1 -ATPase also hydrolyzes ATP at a
decreased rate when VO 2+replaces Mg 2+ [ 21 ] .
The EPR features of VO 2+are able to reveal some details
about how CD39 hydrolyzes ATP and ADP. A single EPR
feature, species T, was observed when ADPNP (a
non-hydrolyzable analog of ATP) complexed with VO 2+was
bound to sCD39, which is consistent with the presence of
only one nucleotide binding site [ 25 ] . The g and A
tensors derived from species T are 1.9410 and 504.25 MHz
respectively, which can be fitted best with one amino group
and three groups combined from carboxyl and phosphate
groups as the equatorial ligands of the bound VO 2+on
sCD39. In accordance with metal-ATP complex coordination on
other enzymes that hydrolyze ATP, like F
1 -ATPase [ 22 28 ] , the γ- and
β-phosphate of ATP most likely bind to VO 2+while the third
carboxyl group is contributed by a side-chain of aspartate
or glutamate of sCD39. It is not unusual for the ε-amino
group of lysine to coordinate with metals in enzymes. It
has been reported that the amino group serves as one of VO
2+equatorial ligands in CF
1 -ATPase [ 21 ] , pyruvate kinase [ 19
20 ] , AdoMet synthetase [ 17 18 ] , and carboxypeptidase [
16 ] . Thus one amino group from lysine, one carboxyl group
from aspartate or glutamate, and two oxygens from the
phosphates of ADPNP serve as the equatorial ligands of
sCD39 bound VO 2+in the presence of ADPNP.
In the presence of AMPCP, bound VO 2+produced two EPR
features, species D1 and species D2 that are separated by
about 30 MHz. As we have reported that sCD39 releases
intermediate ADP before ADP is further cleaved during ATP
hydrolysis [ 25 ] , sCD39 probably has two conformations
that bind metal-ADP complexes, one is the conformation that
releases the ADP intermediate, and another that recruits
intermediate ADP back to the enzyme for further hydrolysis
to AMP. However, intact CD39 does not release intermediate
ADP during ATP hydrolysis, suggesting that there is only
one ADP binding site on each CD39 monomer in the intact
protein [ 2 25 ] . The two EPR species observed with VO
2+-AMPCP probably correspond to the two different
conformations of bound ADP at the same catalytic site on
sCD39. The signal intensity of the bound VO 2+-AMPCP EPR
spectrum indicates that species D1 is dominant over species
D2. In order to further assign species D1 and D2 to the two
different conformations, two experiments were done (Fig.
5). Incubation of sCD39 with VO 2+-AMPCP at room
temperature resulted in a dramatic decrease of the
intensity of species D2, while the signal intensity of
species D1 remained unchanged. These data indicate that VO
2+-AMPCP was released from the conformation corresponding
to species D2; however, the conformation corresponding to
species D1 still had bound VO 2+-AMPCP. More evidence for
two conformations of the enzyme was obtained from the EPR
spectra of bound VO 2+-ATP. No species T was found
presumably because ATP was converted to ADP before the
sample was frozen. Only species D2 was observed and its
intensity decreased as the incubation time was prolonged.
We suggest that species D2 corresponds to the conformation
that releases ADP as an intermediate product and species D1
corresponds to the conformation that binds ADP as a
substrate. The lower signal intensities of species D1 and
D2 compared to that of species T suggest that the affinity
of sCD39 for ADP or its analog AMPCP is lower than that for
the ATP analog ADPNP, which is consistent with the result
that only ATP analogs were detected on sCD39 [ 25 ] .
The calculated g
|| and A
|| values that best matched the
experimental values for species D2 suggest that one
hydroxyl group and three oxygens derived from carboxyl
groups and phosphates are the equatorial ligands of bound
VO 2+-ADP. Since the conformation corresponding to species
D2 is found in the presence of ATP and is likely to be the
conformation that releases bound VO +2-ADP, it is likely
that the VO +2ligands are one phosphate and two carboxyl
groups [ 25 ] . When ADP is the substrate and generates
species D1, one water molecule and a combination of three
groups between carboxyl groups and phosphates serve as the
equatorial ligands of bound VO 2+on sCD39. The probable
combination of carboxyl groups and phosphates for species
D1 is one carboxyl group and two phosphates since VO
2+complexes ADP through two phosphates before VO 2+-ADP is
bound to the enzyme.
The site directed mutagenesis studies on CD39 and other
members of the CD39 family give some hints about the
possible residues that serve as metal ligands at the
catalytic site of sCD39. The changes of D62 on ACR1, E174
on ACR3, D213 (D219 in HB6) and S218 on ACR4 dramatically
decrease both ATPase and ADPase activities of CD39 [ 12 15
] . Figure 7summarizes the possible coordination of Ca
2+from the data of species T, species D1, and species D2 in
the different situations of sCD39 catalysis. The catalytic
base attack results in cleavage of the γ-phosphate of ATP,
and one carboxyl group replaces the γ-phosphate as a metal
ligand (from species T to species D2), which is accompanied
by a swap of an amino group with a hydroxyl group (S218?).
This hydroxyl group (S218?) probably interacts with the
water molecule through hydrogen bond in the conformation
corresponding to species D1 to hydrolyze ADP. The constant
carboxyl group that appears in all conformations of sCD39
hydrolysis is likely contributed by D213 since it is close
to S218.
The results presented here also provide an explanation
to the free metal inhibition of CD39 catalytic activity.
Free VO 2+binds to sCD39 through two hydroxyl groups and
two water molecules that are hydrogen bonded to other
residues of sCD39. Once free VO 2+occupies the catalytic
site, the enzyme has to either release the metal or correct
the conformation before the substrates are recruited
properly.
Conclusions
VO 2+can functionally substitute for Ca 2+as a cofactor
for sCD39. Four different EPR spectra are obtained for VO
2+bound in the presence of different nucleotides and in the
absence of nucleotide. The protein ligands for VO +2in the
presence of ATP are suggested to be carboxyl and amino
groups, while those in the presence of ADP are probably
carboxyl and hydroxyl groups. The mechanism of sCD
catalysis is discussed. These results will provide guides
for further studies of the catalytic mechanism of
NTPDases.
Materials and Methods
Reagents
ATP, ADP, ADPNP, AMPCP were purchased from Sigma (St.
Louis, MO). Zeocin, High-Five medium were purchased from
Invitrogen (Carlsbad, CA).
Cell culture and preparation of soluble CD39
sCD39 transfected stable HighFive™ insect cells were
cultured as described by Chen and Guidotti [ 25 ] .
Soluble CD39 were purified as described [ 25 ] with some
modifications. After concanavalin A-Sepharose 4B and
nickel affinity column chromatography, the ammonium
sulfate precipitated sCD39 was collected and resuspended
in about 50 μl of 40 mM Tris-HCl (pH7.5). This sample was
loaded on a Superose-12HR gel filtration column from
Pharmacia Biotech equilibrated with 40 mM Tris-HCl
(pH7.5). The fractions containing the major peak were
collected, and the solvent was changed to 20 mM Hepes
(pH8.0), 120 mM NaCl, 5 mM KCl with an YM30 centricon
from Millipore. The final volume of the sample was around
200 μl, and the concentration of sCD39 was around 0.1
mM.
Concentrations of proteins were determined using D
C Protein Assay from BIO-RAD using the
provided protocol.
Nucleotidase activity assay and nucleotide
separation by HPLC
The reactions were carried out in 20 mM HEPES-Tris (pH
7.0), 120 mM NaCl, and 5 mM KCl; they were started by
adding nucleotides at 37°C. After incubation for 15
minutes, the reactions were stopped with 2%
perchloroacetic acid
Nucleotides were separated by HPLC on an anion
exchange column (a 10 × 0.46 mm SAX column from Rainin
Instruments) based on the method of Hartwick and Brown [
29 ] . The low concentration buffer (A) was 0.08 M NH
4 H
2 PO
4 (pH3.8), and the high concentration
buffer (B) was 0.25 M NH
4 H
2 PO
4 (pH4.95) with 8 mM KCl. The gradient
used was 4 min, 0-2.5% (B); 26 min, 2.5-25% (B).
Equilibration was done with buffer (A) for 10 minutes,
and the flow rate was 1 ml/min.
Preparation of VO 2+solution
Vanadyl and nucleotide solution were prepared
according to Houseman et al. [ 21 ] . Dissolved molecular
oxygen was removed from solutions by purging with dry
nitrogen gas. Stock vanadyl and nucleotide solution were
thawed on ice, and mixed at 1:1 molar ratio by vigorous
stirring. Then VO 2+-nucleotide complexes were added to
purified sCD39 at 1:1 molar ratio, mixed, and incubated
for 5 minutes on ice before they were transferred into
EPR tubes. Once the samples were in EPR tubes, they were
immediately frozen in liquid nitrogen, and stored in
liquid nitrogen before using.
EPR Measurement
CW-EPR experiments were carried out at X-band (9 GHz)
using a Bruker 300E spectrometer with a TE102 rectangular
standard cavity and a liquid nitrogen flow cryostat
operating at 150 K. Simulations of these EPR spectra were
accomplished with the computer program QPOWA [ 30 31 ]
).
To estimate the types of groups that serve as
equatorial ligands to VO 2+in each condition, the
observed values of A
|| derived from simulation of the EPR
spectrum by QPOWA were compared with the coupling
constants obtained from model studies [ 24 32 ]
using:
A
||calc = Σ n
i A
||i /4
where i represents the different types of equatorial
ligand donor groups, n
i (=1-4) is the number of ligands of
type i, and A
||i is the measured coupling constant
for equatorial donor group i [ 24 ] . Similar equations
were used to calculated g
|| from a given set of equatorial
ligands for comparison with those derived
experimentally.
