- Research article
- Open Access
The Metal Coordination of sCD39 during ATP Hydrolysis
© Chen and Guidotti; licensee BioMed Central Ltd. 2001
- Received: 25 July 2001
- Accepted: 12 September 2001
- Published: 12 September 2001
The hydrolysis of ATP and ADP by ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) requires divalent cations, like Ca2+ and Mg2+. In spite of considerable work, it is not clear whether divalent cations bind to the enzyme in the absence of nucleotide or only as nucleotide-Me+2 complex. Here we study the protein ligands for Me+2.
When VO2+ was used as a substitute for Ca2+, the ATPase activity of soluble CD39 was 25% of that with Ca2+ as cofactor. Protein ligands of the VO2+-nucleotide complex bound to the catalytic site of soluble CD39 were characterized by electron paramagnetic resonance (EPR) spectroscopy. The EPR spectrum contained one species designated T with VO2+-AMPPNP as ligand. Two species D1 and D2 were observed when VO2+-AMPCP was bound to soluble CD39. The results suggest that species D1 and D2 represent the metal-ADP complexes at the catalytic site of soluble CD39 corresponding to the intermediate formed during ATP hydrolysis and the substrate for further hydrolysis, respectively.
VO2+ can functionally substitute for Ca2+ as a cofactor of sCD39, and it produces four different EPR features when bound in the presence of different nucleotides or in the absence of nucleotide. The metal coordination for each conformation corresponding to each EPR species is proposed, and the mechanism of sCD39 catalysis is discussed.
- Electron Paramagnetic Resonance
- Electron Paramagnetic Resonance Spectrum
- Soluble CD39
- Nucleotidase Activity
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 . Three isoforms that differ in the ratio of ATPase/ADPase activity are present on the cell surface : 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 . 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 [3–6]. The catalytic reaction appears to be irreversible and no partial reactions have been observed [7, 8]. Divalent cations like Ca2+ or Mg2+ are required for activity, and maximal activity is reached when the concentrations of substrates and divalent cations are equal . 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 .
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 . Substitution of H59 in ACR1 converted CD39 into an ADPase in a quaternary structure dependent manner . Mutation of W187A in ACR3 affected CD39 folding and translocation, while mutation of W459A in ACR5 increased ATPase activity but diminished ADPase activity . 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 . 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+2 and Mg+2 with proteins are difficult to study due to the lack of spectroscopic properties, vanadyl (VIV=O)2+ has been used as a probe of the ligands that compose Mg2+, Ca2+, and Mn2+ binding sites of several proteins, including carboxypeptidase , S-adenosylmethionine synthetase [17, 18], pyruvate kinase [19, 20], and F1-ATPase [21, 22]. This cation specifically binds to divalent cation binding sites of several enzymes, and in many cases serves as a functional cofactor . 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 Ca2+ and Mg2+. As it is known that the A and g tensors derived from the EPR spectrum of bound VO2+ are a direct measure of the nature of the equatorial metal ligands , binding of VO2+ 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 . Only one nucleotide-binding site was identified on the purified soluble CD39 in the presence of Ca2+ when non-hydrolysable nucleotide analogs were used. In this report, we characterized the signals that were obtained from bound VO2+ when ATP or ADP was present at the catalytic site of the purified soluble CD39. The possible metal ligands for VO+2 at the catalytic site are proposed and the catalytic mechanism is discussed.
Nucleotidase activity of purified soluble CD39 with VO2+ as cofactor
Characterization of bound VO2+ ADPNP by CW-EPR
Experimental signal intensity and 51 V-hyperfine parameters derived from the VO2+ bound to sCD39 under different conditions.
(% of VOADPNP)
Best fits of the 51 V-hyperfine parameters (Eq. 1) of VO2+ bound to various equatorial ligands of sCD39 under different conditions.
Most Probable Equatorial Ligands
Characterization of EPR species from VO2+-AMPCP bound to sCD39
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 VO2+-ATP
The same sample made from mixing VO2+-ATP and sCD39 was incubated at room temperature for 30 minutes, then the VO2+-EPR spectrum was generated as shown in Figures 4c and 4d. The EPR parameters derived from this VO2+-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 VO2+ binding to sCD39 characterized by CD-EPR
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.
Vanadyl has been used to estimate the types of groups that serve as metal-ligands in F1-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 VO2+ 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 VO2+ can functionally replace Ca2+ in the hydrolysis of both ATP and ADP by soluble CD39, although the enzymatic activity is about 25% of that with Ca2+ as the cofactor, since F1-ATPase also hydrolyzes ATP at a decreased rate when VO2+ replaces Mg2+.
The EPR features of VO2+ 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 VO2+ was bound to sCD39, which is consistent with the presence of only one nucleotide binding site . 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 VO2+ on sCD39. In accordance with metal-ATP complex coordination on other enzymes that hydrolyze ATP, like F1-ATPase [22, 28], the γ- and β-phosphate of ATP most likely bind to VO2+ 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 VO2+ equatorial ligands in CF1-ATPase , pyruvate kinase [19, 20], AdoMet synthetase [17, 18], and carboxypeptidase . 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 VO2+ in the presence of ADPNP.
In the presence of AMPCP, bound VO2+ 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 , 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 VO2+-AMPCP probably correspond to the two different conformations of bound ADP at the same catalytic site on sCD39. The signal intensity of the bound VO2+-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 VO2+-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 VO2+-AMPCP was released from the conformation corresponding to species D2; however, the conformation corresponding to species D1 still had bound VO2+-AMPCP. More evidence for two conformations of the enzyme was obtained from the EPR spectra of bound VO2+-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 .
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 VO2+-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+2 ligands are one phosphate and two carboxyl groups . 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 VO2+ on sCD39. The probable combination of carboxyl groups and phosphates for species D1 is one carboxyl group and two phosphates since VO2+ complexes ADP through two phosphates before VO2+-ADP is bound to the enzyme.
The results presented here also provide an explanation to the free metal inhibition of CD39 catalytic activity. Free VO2+ binds to sCD39 through two hydroxyl groups and two water molecules that are hydrogen bonded to other residues of sCD39. Once free VO2+ occupies the catalytic site, the enzyme has to either release the metal or correct the conformation before the substrates are recruited properly.
VO2+ can functionally substitute for Ca2+ as a cofactor for sCD39. Four different EPR spectra are obtained for VO2+ bound in the presence of different nucleotides and in the absence of nucleotide. The protein ligands for VO+2 in 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.
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 . Soluble CD39 were purified as described  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 DC 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 . The low concentration buffer (A) was 0.08 M NH4H2PO4 (pH3.8), and the high concentration buffer (B) was 0.25 M NH4H2PO4 (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 VO2+ solution
Vanadyl and nucleotide solution were prepared according to Houseman et al. . 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 VO2+-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.
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 VO2+ 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 niA||i/4
where i represents the different types of equatorial ligand donor groups, ni (=1–4) is the number of ligands of type i, and A||i is the measured coupling constant for equatorial donor group i . Similar equations were used to calculated g|| from a given set of equatorial ligands for comparison with those derived experimentally.
This work was supported by Grant HL08893 from the National Institutes of Health.
- Plesner L: Ecto-ATPases: identities and functions. Int Rev Cytol. 1995, 158: 141-214.View ArticlePubMedGoogle Scholar
- Zimmermann H, Braun N, Heine P, Kohring K, Marxen M, Sevigny J, Robson SC: The molecular and functional properties of E-NTPDase1, E-NTPDase2 and Ecto-5' nucleotidase in nervous tissue. Proceedings of the Second International Workshop on Ecto-ATPase and Related Ectonucleotidases. 2000, 9-20.Google Scholar
- Martin SS, Senior AE: Membrane adenosine triphosphatase activities in rat pancreas. Biochim Biophys Acta. 1980, 602: 401-18.View ArticlePubMedGoogle Scholar
- Hidalgo C, Gonzalez ME, Lagos R: Characterization of the Ca2+- or Mg2+-ATPase of transverse tubule membranes isolated from rabbit skeletal muscle. J Biol Chem. 1983, 258: 13937-45.PubMedGoogle Scholar
- Obejero Paz CA, Gonzalez DA, Alonso GL: Demonstration of the simultaneous activation of Ca2+-independent and Ca2+-dependent ATPases from rat skeletal muscle microsomes. Biochim Biophys Acta. 1988, 939: 409-15.View ArticlePubMedGoogle Scholar
- Valente AP, Barrabin H, Jorge RV, Paes MC, Scofano HM: Isolation and characterization of the Mg2(+)-ATPase from rabbit skeletal muscle sarcoplasmic reticulum membrane preparations. Biochim Biophys Acta. 1990, 1039: 297-304.View ArticlePubMedGoogle Scholar
- Norton K, Moulton M, Rose R, Sabbadini R, Dahms AS: Biophys. J. 1986, 49: 561a-Google Scholar
- Sabbadini RA, Dahms AS: Biochemical properties of isolated transverse tubular membranes. J Bioenerg Biomembr. 1989, 21: 163-213.View ArticlePubMedGoogle Scholar
- Handa M, Guidotti G: Purification and cloning of a soluble ATP-diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum). Biochem Biophys Res Commun. 1996, 218: 916-23. 10.1006/bbrc.1996.0162.View ArticlePubMedGoogle Scholar
- Stout JG, Kirley TL: Purification and characterization of the ecto-Mg-ATPase of chicken gizzard smooth muscle. J Biochem Biophys Methods. 1994, 29: 61-75.View ArticlePubMedGoogle Scholar
- Schulte am Esch J, Sevigny J, Kaczmarek E, Siegel JB, Imai M, Koziak K, Beaudoin AR, Robson SC: Structural elements and limited proteolysis of CD39 influence ATP diphosphohydrolase activity. Biochemistry. 1999, 38: 2248-58. 10.1021/bi982426k.View ArticlePubMedGoogle Scholar
- Drosopoulos JHF, Broekman MJ, Islam N, Maliszewski CR, Gayle RB, Marcus AJ: Site-directed mutagenesis of human endothelial cell ecto-ADPase/soluble CD39: requirement of glutamate 174 and serine 218 for enzyme activity and inhibition of platelet recruitment. Biochemistry. 2000, 39: 6936-43. 10.1021/bi992581e.View ArticlePubMedGoogle Scholar
- Grinthal A, Guidotti G: Substitution of His59 converts CD39 apyrase into an ADPase in a quaternary structure dependent manne. Biochemistry. 2000, 39: 9-16. 10.1021/bi991751k.View ArticlePubMedGoogle Scholar
- Smith TM, Lewis Carl SA, Kirley TL: Mutagenesis of two conserved tryptophan residues of the E-type ATPases: inactivation and conversion of an ecto-apyrase to an ecto-NTPase. Biochemistry. 1999, 38: 5849-57. 10.1021/bi990171k.View ArticlePubMedGoogle Scholar
- Smith TM, Kirley TL: Site-directed mutagenesis of a human brain ecto-apyrase: evidence that the E-type ATPases are related to the actin/heat shock 70/sugar kinase superfamily. Biochemistry. 1999, 38: 321-8. 10.1021/bi9820457.View ArticlePubMedGoogle Scholar
- DeKoch RJ, West DJ, Cannon JC, Chasteen ND: Kinetics and electron paramagnetic resonance spectra of vanadyl(IV) carboxypeptidase A. Biochemistry. 1974, 13: 4347-54.View ArticlePubMedGoogle Scholar
- Markham GD: Structure of the divalent metal ion activator binding site of S-adenosylmethionine synthetase studied by vanadyl(IV) electron paramagnetic resonance. Biochemistry. 1984, 23: 470-8.View ArticlePubMedGoogle Scholar
- Zhang C, Markham GD, LoBrutto R: Coordination of vanadyl(IV) cation in complexes of S-adenosylmethionine synthetase: multifrequency electron spin echo envelope modulation study. Biochemistry. 1993, 32: 9866-73.View ArticlePubMedGoogle Scholar
- Tipton PA, McCracken J, Cornelius JB, Peisach J: Electron spin echo envelope modulation studies of pyruvate kinase active-site complexes. Biochemistry. 1989, 28: 5720-8.View ArticlePubMedGoogle Scholar
- Lord K, Reed GH: Vanadyl(IV) complexes with pyruvate kinase: activation of the enzyme and electron paramagnetic resonance properties of ternary complexes with the protein. Arch Biochem Biophys. 1990, 281: 124-31.View ArticlePubMedGoogle Scholar
- Houseman AL, Morgan L, LoBrutto R, Frasch WD: Characterization of ligands of a high-affinity metal-binding site in the latent chloroplast F1-ATPase by EPR spectroscopy of bound VO2+. Biochemistry. 1994, 33: 4910-7.View ArticlePubMedGoogle Scholar
- Chen W, LoBrutto R, Frasch WD: EPR spectroscopy of VO2+-ATP bound to catalytic site 3 of chloroplast F1-ATPase from Chlamydomonas reveals changes in metal ligation resulting from mutations to the phosphate-binding loop threonine (betaT168). J Biol Chem. 1999, 274: 7089-94.View ArticlePubMedGoogle Scholar
- Eaton SS, Eaton GR: Vanadium in Biological Systems (Chasteen, N. D., Ed). Kluwer Academic Publishers, Dordrecht, The Netherlands. 1990, 199-222.Google Scholar
- Chasteen ND: Biological Magnetic Resonance (Berliner, L., and Reuben, J., Eds). Plenum, New York. 1990, 53-119.Google Scholar
- Chen W, Guidotti G: Soluble apyrases release ADP during ATP hydrolysis. Biochem Biophys Res Commun. 2001, 282: 90-5. 10.1006/bbrc.2001.4555.View ArticlePubMedGoogle Scholar
- Weil JA, Hecht AG: On the powder line shape of EPR spectra. J Chem Phys. 1963, 38: 281-286.View ArticleGoogle Scholar
- Holyk N: M. S. Thesis,. University of New Hampshire, Durham, NH. 1979Google Scholar
- Houseman AL, LoBrutto R, Frasch WD: Effects of nucleotides on the protein ligands to metals at the M2 and M3 metal-binding sites of the spinach chloroplast F1-ATPase. Biochemistry. 1995, 34: 3277-85.View ArticlePubMedGoogle Scholar
- Hartwick RA, Brown PR: The use of high pressure liquid chromatography in clinical chemistry and biomedical research. Adv Clin Chem. 1980, 21: 25-99.View ArticlePubMedGoogle Scholar
- Nilges MJ: Electron Paramagnetic Resonance Studies of Low Symmetry Nickel (I) and Molybenum (V) Complexes, Ph.D. Thesis,. University of Illinois, Urbana, IL. 1979Google Scholar
- Maurice MJ: Acquisition of Anisotropic Information by Computational Analysis of Isotropic EPR Spectra, Ph. D. Thesis,. University of Illinois, Urbana, IL. 1980Google Scholar
- Hamstra BJ, Houseman AL, Colpas GL, Kampf JW, LoBrutto R, Frasch WD, Pecoraro VL: Structural and solution characterization of mononuclear vanadium (IV) complexes that help to elucidate the active site structure of the reduced vanadium haloperoxidases. Inorg Chem. 1997, 36: 4866-4874. 10.1021/ic970284x.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. Verbatim copying and redistribution of this article are permitted in any medium for any non-commercial purpose, provided this notice is preserved along with the article's original URL. For commercial use, contact email@example.com