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• W2079723980 abstract "In mammalian cells, there are three pyrimidine nucleoside salvage enzymes with the capacity to phosphorylate all four deoxynucleosides, the two thymidine kinase isoenzymes, TK1 and TK2, and the deoxycytidine kinase, dCK. TK1 is cell cycle-regulated; TK2 is expressed constitutively and can phosphorylate deoxycytidine to the same extent as thymidine. dCK phosphorylates deoxycytidine, deoxyadenosine, and deoxyguanosine, but not thymidine. In addition, the three kinases can phosphorylate a number of medically important analogs. In cultured Drosophila melanogaster embryonic cells, only one pyrimidine deoxynucleoside kinase was present. This kinase was purified and showed a broad substrate specificity, since it was able to phosphorylate all four deoxynucleosides with high efficiency, as compared with the kinases in mammalian cells. Additionally, a number of nucleoside analogs such as arabinofuranosyl pyrimidines, deoxyuridine, and 5′-fluorodeoxyuridine, were phosphorylated. There was negligible 3′-azidothymidine and no dTMP phosphorylation. The enzyme was active as a monomer of about 30 kDa. We suggest the name D. melanogaster deoxynucleoside kinase for this multifunctional kinase. The substrate specificity, size, and other characteristics show that this enzyme is more related to human TK2 than to the other mammalian deoxyribonucleoside kinases, but is unique with respect to the capacity to phosphorylate all four deoxynucleosides. In mammalian cells, there are three pyrimidine nucleoside salvage enzymes with the capacity to phosphorylate all four deoxynucleosides, the two thymidine kinase isoenzymes, TK1 and TK2, and the deoxycytidine kinase, dCK. TK1 is cell cycle-regulated; TK2 is expressed constitutively and can phosphorylate deoxycytidine to the same extent as thymidine. dCK phosphorylates deoxycytidine, deoxyadenosine, and deoxyguanosine, but not thymidine. In addition, the three kinases can phosphorylate a number of medically important analogs. In cultured Drosophila melanogaster embryonic cells, only one pyrimidine deoxynucleoside kinase was present. This kinase was purified and showed a broad substrate specificity, since it was able to phosphorylate all four deoxynucleosides with high efficiency, as compared with the kinases in mammalian cells. Additionally, a number of nucleoside analogs such as arabinofuranosyl pyrimidines, deoxyuridine, and 5′-fluorodeoxyuridine, were phosphorylated. There was negligible 3′-azidothymidine and no dTMP phosphorylation. The enzyme was active as a monomer of about 30 kDa. We suggest the name D. melanogaster deoxynucleoside kinase for this multifunctional kinase. The substrate specificity, size, and other characteristics show that this enzyme is more related to human TK2 than to the other mammalian deoxyribonucleoside kinases, but is unique with respect to the capacity to phosphorylate all four deoxynucleosides. The biosynthesis of deoxynucleoside triphosphates in most living organisms is performed by two pathways, the de novo and the salvage pathway. The main enzyme of the de novo pathway is ribonucleotide reductase, which catalyzes the reduction of the 2′-OH group of the nucleoside diphosphates. The enzyme has been extensively studied as to the structure and the complex feedback regulation by the end products, the four deoxynucleoside triphosphates (1Reichard P. Annu. Rev. Biochem. 1988; 57: 349-374Crossref PubMed Scopus (625) Google Scholar, 2Reichard P. Ehrenberg A. Science. 1983; 221: 514-519Crossref PubMed Scopus (383) Google Scholar).An alternative pathway for supplying deoxynucleoside triphosphates for replication and repair of DNA is the salvage pathway. The principal salvage enzymes are the deoxynucleoside kinases, which phosphorylate deoxynucleosides to the corresponding deoxynucleoside monophosphates. In mammalian cells, there are four deoxynucleoside kinases with different cellular localization and specificities, thymidine kinase 1 (TK1), 1The abbreviations used are: TK1, cytoplasmic specific thymidine kinase; TK2, mitochondrial thymidine kinase; AraC, 1-β-d-arabinofuranosylcytosine; AraT, 1-β-d-arabinofuranosylthymine; AZT, 3′-azido-2′,3′-dideoxythymidine; CHAPS, 3-((3-cholamidopropyl)dimethylammonio)-1-propylsulfonic acid; dAdo, deoxyadenosine; dAK, deoxyadenosine kinase; dCK, deoxycytidine kinase; dCyd, deoxycytidine; dGK, deoxyguanosine kinase; dGuo, deoxyguanosine; Dm-dNK, D. melanogasterdeoxynucleoside kinase; dThd, thymidine; dUrd, deoxyuridine; FddThd, 3′-fluoro-2′,3′-dideoxythymidine; FdUrd, 5-fluorodeoxyuridine; TK, thymidine kinase; CV, coefficient of variation.1The abbreviations used are: TK1, cytoplasmic specific thymidine kinase; TK2, mitochondrial thymidine kinase; AraC, 1-β-d-arabinofuranosylcytosine; AraT, 1-β-d-arabinofuranosylthymine; AZT, 3′-azido-2′,3′-dideoxythymidine; CHAPS, 3-((3-cholamidopropyl)dimethylammonio)-1-propylsulfonic acid; dAdo, deoxyadenosine; dAK, deoxyadenosine kinase; dCK, deoxycytidine kinase; dCyd, deoxycytidine; dGK, deoxyguanosine kinase; dGuo, deoxyguanosine; Dm-dNK, D. melanogasterdeoxynucleoside kinase; dThd, thymidine; dUrd, deoxyuridine; FddThd, 3′-fluoro-2′,3′-dideoxythymidine; FdUrd, 5-fluorodeoxyuridine; TK, thymidine kinase; CV, coefficient of variation. thymidine kinase 2 (TK2), deoxycytidine kinase (dCK), and deoxyguanosine kinase (dGK). TK1 is cytosolic and is expressed only in S-phase cells (3Sherley J.L. Kelly T.J. J. Biol. Chem. 1988; 263: 8350-8358Abstract Full Text PDF PubMed Google Scholar, 4Kauffman M.G. Kelly T.J. Mol. Cell. Biol. 1991; 11: 2538-2546Crossref PubMed Scopus (111) Google Scholar); the corresponding gene was cloned several years ago (5Bradshaw Jr., H.D. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5588-5591Crossref PubMed Scopus (50) Google Scholar). TK2, dCK, and dGK are all constitutively expressed enzymes. TK2 is considered to be localized in mitochondria (6Kit S. Leung W.-C. Biochem. Genet. 1974; 11: 231-247Crossref PubMed Scopus (32) Google Scholar), but is encoded by a nuclear gene that was cloned recently (7Johansson M. Karlsson A. J. Biol. Chem. 1997; 272: 8454-8458Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). dCK is cytosolic, and the corresponding gene was cloned in 1991 (8Chottiner E.G. Shewach D.S. Datta N.S. Ashcraft E. Gribbin D. Ginsburg D. Fox I.H. Mitchell B.S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1531-1535Crossref PubMed Scopus (135) Google Scholar). dGK is mitochondrial (9Gower Jr., W.J. Carr M.C. Ives D.H. J. Biol. Chem. 1979; 254: 2180-2183Abstract Full Text PDF PubMed Google Scholar) and has a manyfold lower phosphorylation capacity compared with the other three pyrimidine nucleoside kinases. Like TK2, dGK is encoded by a nuclear gene, which has recently been cloned by two independent groups (10Wang L. Hellman U. Eriksson S. FEBS Lett. 1996; 390: 39-43Crossref PubMed Scopus (55) Google Scholar, 11Johansson M. Karlsson A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7258-7262Crossref PubMed Scopus (81) Google Scholar). Homology studies indicate that mammalian TK1 is closely related to pox-viral TKs and TK from Escherichia coli (12Black M.E. Hruby D.E. Mol. Microbiol. 1991; 5: 373-379Crossref PubMed Scopus (20) Google Scholar), whereas dCK, dGK, and TK2 share many properties with herpetic TKs (7Johansson M. Karlsson A. J. Biol. Chem. 1997; 272: 8454-8458Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The substrate specificities of the mammalian deoxynucleoside kinases toward natural substrates and a number of therapeutic nucleoside analogs differ considerably, but with a characteristic pattern useful for distinction between the enzymes (13Arnér E.S.J. Spasokukotskaja T. Eriksson S. Biochem. Biophys. Res. Commun. 1992; 188: 712-718Crossref PubMed Scopus (146) Google Scholar). In addition to thymidine (dThd), 2′-deoxyuridine (dUrd), and 5-fluoro-2′-deoxyuridine (FdUrd), TK1 can phosphorylate 3′-azidothymidine (AZT) and 3′-fluoro-2′,3′-dThd (FddThd), and TK2 can phosphorylate deoxycytidine (dCyd) and 1-β-d-arabinofuranosyl thymine (AraT) (13Arnér E.S.J. Spasokukotskaja T. Eriksson S. Biochem. Biophys. Res. Commun. 1992; 188: 712-718Crossref PubMed Scopus (146) Google Scholar, 14Munch-Petersen B. Cloos L. Tyrsted G. Eriksson S. J. Biol. Chem. 1991; 266: 9032-9038Abstract Full Text PDF PubMed Google Scholar). dCK can phosphorylate deoxyadenosine (dAdo), deoxyguanosine (dGuo) and 1-β-d-arabinofuranosyl cytosine (AraC) (15Bohman C. Eriksson S. Biochemistry. 1988; 27: 4258-4265Crossref PubMed Scopus (98) Google Scholar), and dGK can phosphorylate dAdo (16Wang L. Karlsson A. Arnér E.S.J. Eriksson S. J. Biol. Chem. 1993; 268: 22847-22852Abstract Full Text PDF PubMed Google Scholar).The occurrence of cytosolic and mitochondrial forms of thymidine kinases has also been observed in lower eucaryotes. In the filamentous fungus Achlya ambisexualis, four forms of TK were found, one readily solubilized and three solubilized by detergent. The multiple TK forms were distinctive by their electrophoretic properties, and one of the detergent solubilized forms resembled TK2 in the ability to phosphorylate dCyd (17Lam F.K. Jing G.J. Leung W.-C. Biochem. Cell. Biol. 1988; 66: 318-324Crossref Google Scholar).Drosophila melanogaster, the fruit fly, is one of the genetically and developmentally most characterized organisms. However, so far, the metabolism of deoxynucleotides and its regulation and role in the cell proliferation and differentiation during embryogenesis has been only poorly investigated, and to our knowledge there have been no reports about deoxynucleoside kinases in this organism or other insect cells. In the present paper, we describe the purification and characterization of a deoxynucleoside kinase from the fruit fly. The purified kinase is multifunctional and apparently the only deoxynucleoside kinase present in the cultured DrosophilaS-2 cells.EXPERIMENTAL PROCEDURESMaterialsDEAE-Sepharose Fast Flow, Phenyl-Sepharose, the Superose 12 HR 10/30 column, and SDS-polyacrylamide molecular weight standards were from Pharmacia Biotech Inc. The 3′-dTMP-Sepharose gel-matrix (p-aminophenyl-3′-TMP:CH-Sepharose) was prepared according to the procedure described by Kowal and Markus (18Kowal E.P. Markus G. Prep. Biochem. 1976; 6: 369-385PubMed Google Scholar), using thymidine-3′-(4-aminophenyl-phosphate) kindly donated by Eger et al. (19Eger K. Klunder E. Beck R.A. Schloz U. Pharmaceut. Res. 1993; 10: 771-773Crossref PubMed Scopus (5) Google Scholar). Fetal calf serum was purchased from Life Technologies, Inc., and the gel-filtration molecular weight standards, Schneider's medium, unlabeled nucleotides and nucleosides, ultrapure ammonium sulfate and CHAPS were purchased from Sigma. 3H-Labeled thymidine (925 GBq/mmol) and deoxycytidine (740–925 GBq/mmol) were obtained from Amersham Corp. 3H-Labeled deoxyadenosine (1106 GBq), deoxyguanosine (226 GBq/mmol), deoxyuridine (629 GBq/mmol), 5-fluoro-deoxyuridine (703 GBq), 2′,3′-dideoxy-2′,3′-didehydrothymidine (744 GBq/mmol), 3′-fluoro-2′,3′-dideoxythymidine (289 GBq/mmol), 3′-azido-2′,3′-dideoxythymidine (740 GBq/mmol), 1-β-d-arabinofuranosyl thymine (107 GBq/mmol), 1-β-d-arabinofuranosyl cytosine (862 GBq/mmol), 2′,3′-dideoxycytidine (185 GBq/mmol), and acyclovir (655 GBq/mmol) were from Moravek Biochemicals Inc., Brea, CA. When present, ethanol was evaporated and the analog resuspended in water. All other chemicals were of the highest purity available. All solutions were made with ultrafiltered (0.22 μm) and autoclaved water, purified by the Milli-RO 15 water system from Millipore.CellsThe D. melanogaster S-2 cell line of embryonic origin were grown in Schneider's medium supplemented with 10% fetal calf serum in a Brown Biostat M bioreactor (B. Brown, Melsungen, Germany) as described previously (20Søndergaard L. Biotechnol. Tech. 1996; 10: 161-166Crossref Google Scholar). Cells (8.6 × 1010) were harvested by centrifugation (2000 × g) and pellets stored at −80 °C until used for enzyme purification.Enzyme AssaysThe nucleoside kinase activities were determined by initial velocity measurements based on four time samples by the DE-81 filter paper assay using tritium-labeled substrates (14Munch-Petersen B. Cloos L. Tyrsted G. Eriksson S. J. Biol. Chem. 1991; 266: 9032-9038Abstract Full Text PDF PubMed Google Scholar). The standard assay conditions were: 50 mm Tris-HCl, pH 8.0 (22 °C), 2.5 mm MgCl2, 10 mm dithiothreitol, 0.5 mm CHAPS, 3 mg/ml bovine serum albumin, 2.5 mmATP, and 10 μm radiolabeled substrate, unless otherwise indicated.The products of the kinase reaction were analyzed by mixing samples of 10 μl from a standard reaction mixture with 20 μl of 5 mm dThd, dTMP, dTDP, and dTTP, and spotting 10 μl of this solution on polyethyleneimine-cellulose plates. After developing the plates ascending in 0.5 m LiCl2, the spots with nucleosides and nucleotides were identified under UV light and cut out. The radioactivity was extracted with 0.2 m KCl, 0.1m HCl and determined by liquid scintillation.The variance in duplicate assays was below 5% (CV). The variance in the determination of kinetic constants in two independent experiments was below 20% (CV). One unit of activity is defined as 1 nmol of deoxynucleoside 5′-monophosphate formed/min. Specific activity is expressed as units/mg of protein, where the kinase activity is measured at saturating substrate concentrations.Enzyme PurificationBuffersBuffer A consisted of 20 mm potassium phosphate buffer (pH 7.4), 15% glycerol, 1 mm potassium EDTA, 1 mm dithiothreitol. Buffer B consisted of 20 mm Tris, pH 7.5 (20 °C), 5 mmMgCl2, 10% glycerol, 2 mm dithiothreitol. Buffer C consisted of 10 mm Tris-HCl, pH 8 (20 °C), 5 mm MgCl2, 10% glycerol, 2 mmdithiothreitol, 0.5 mm CHAPS. Buffer D consisted of 50 mm Tris-HCl, pH 7.5 (20 °C), 5 mmMgCl2, 10% glycerol, 2 mm dithiothreitol, 0.5 mm CHAPS. Buffer E consisted of 50 mm potassium phosphate buffer, pH 7.5, 5 mm MgCl2, 10% glycerol, 2 mm dithiothreitol. Buffer F consisted of 50 mm imidazole-HCl, pH 7.5, 5 mmMgCl2, 2 mm dithiothreitol, 0.1 mKCl.Purification steps I–V were performed at 4 °C according to previously described procedures (14Munch-Petersen B. Cloos L. Tyrsted G. Eriksson S. J. Biol. Chem. 1991; 266: 9032-9038Abstract Full Text PDF PubMed Google Scholar) with minor modifications.Step I: Preparation of Crude ExtractThe pellet containing 8.6 × 1010 Drosophila S-2 cells was suspended in buffer A and homogenized by treatment with a French press. The homogenate was centrifuged 30 min at 12,000 × g(fraction I).Step II: Streptomycin/Ammonium Sulfate Precipitation and G-25 ChromatographyThe nucleic acids were precipitated with streptomycin sulfate (0.7%), and the resulting supernatant precipitated with ammonium sulfate in two steps (20% and 70%) as described elsewhere (15Bohman C. Eriksson S. Biochemistry. 1988; 27: 4258-4265Crossref PubMed Scopus (98) Google Scholar). All centrifugations were performed at 12,000 × g. The ammonium sulfate pellet was suspended in buffer B, divided in three portions of about 30 ml, and each portion was desalted on a Sephadex G-25 column (bed volume, 50 mm × 200 mm) with buffer B. The peak fractions were collected and pooled (fraction II).Step III: DEAE Ion Exchange ChromatographyFraction II was divided in three portions, and each portion was chromatographed on a DEAE Sepharose Fast Flow column (bed volume, 50 mm × 250 mm), equilibrated with buffer B + 0.5 mm CHAPS. After application, unbound material was washed out with buffer B + 0.5 mm CHAPS (600 ml) and the bound material was eluted with a 0–0.5 m KCl gradient in buffer B (2 × 1.2 liters). The fractions containing TK and dCK activity were pooled (fraction III).Step IV: 3′-dTMP-Sepharose ChromatographyFraction III was divided in three portions, and each portion was applied on a 3′-dTMP-Sepharose column (bed volume, 10 mm × 64 mm) equilibrated with buffer C. The unbound material was washed out with buffer C and then with buffer D, and the bound material was eluted with buffer D containing 2 mm dThd. The fractions containing TK activity were pooled (fraction IV).Step V: Phenyl-Sepharose ChromatographyFraction IV was made 1.5 m with ammonium sulfate and applied on a Phenyl-Sepharose column (10 mm × 12.8 mm) equilibrated with buffer E + 1.5 m ammonium sulfate. The unbound material was washed out with the equilibration buffer. The bound material was eluted in two steps, first with buffer E and thereafter with buffer E + 10 mm CHAPS.Molecular Weight DeterminationsThe subunit size of the purified protein was determined by discontinuous SDS-polyacrylamide gel electrophoresis in Tris-HCl (pH 6.8 and 8.8, 22 °C) with a stacking gel of 4.5% and a separating gel of 12%, according to the procedure of Laemmli (21Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206024) Google Scholar). The protein bands were visualized by silver staining.The apparent size of the native enzyme was determined by gel-filtration on a prepacked Superose 12 column connected to a Gradifrac system essentially as described (14Munch-Petersen B. Cloos L. Tyrsted G. Eriksson S. J. Biol. Chem. 1991; 266: 9032-9038Abstract Full Text PDF PubMed Google Scholar). The column was equilibrated and the enzyme eluted with buffer F.Other MethodsThe protein concentration was determined according to Bradford (22Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213377) Google Scholar). Conductivity was measured with the CDM 3 Radiometer conductivity meter using the CDC 314 cell.DISCUSSIONThe present work was initiated with the purpose to investigate the pattern of TK isoenzymes in D. melanogaster, and to compare it with the situation in other organisms. In the initial purification steps, the TK isoenzymes from mammalian cells, TK1 and TK2, were separated from each other and from dCK by DEAE ion exchange chromatography (14Munch-Petersen B. Cloos L. Tyrsted G. Eriksson S. J. Biol. Chem. 1991; 266: 9032-9038Abstract Full Text PDF PubMed Google Scholar). When the desalted ammonium sulfate fraction from cultured Drosophila S-2 cells (fraction II) was chromatographed on DEAE-Sepharose under conditions identical to those applied for the mammalian enzymes, only one peak of TK and dCK activity was obtained, with the activities coinciding (Fig. 1). This indicated the absence of deoxynucleoside kinases equivalent to the mammalian TK1 and dCK.The single peak obtained from the DEAE chromatography contained all four deoxynucleoside kinase activities, and these activities were co-purified more than 20,000-fold (Table I) to nearly 99% purity, as judged from SDS-gel electrophoresis (Fig. 2). The activities were not separable during the molecular weight determinations. In the SDS gel, there was a single band with a mass of 30 kDa (Fig. 2), and the four kinase activities migrated together on the Superose 12 column with an apparent size of about 33 kDa (Fig. 3). Furthermore, the dAK, dCK, dGK, and TK activities were all bound to the dTMP-Sepharose matrix, were eluted together in the thymidine buffer, and were bound to Phenyl-Sepharose, where the strong binding required more than 5 mm CHAPS to elute the activities. Together, these results indicate that the four deoxynucleoside kinase activities were associated to the same monomeric protein of about 30 kDa. It is proposed to designate this multifunctional kinase asDm-dNK.The occurrence of multifunctional deoxynucleoside kinases with two or three of the four deoxynucleoside kinase activities has been observed previously in mammalian cells. TK2 is also a dCyd kinase (14Munch-Petersen B. Cloos L. Tyrsted G. Eriksson S. J. Biol. Chem. 1991; 266: 9032-9038Abstract Full Text PDF PubMed Google Scholar), and dCK can phosphorylate dAdo and dGuo, although with lower efficiency than dCyd (15Bohman C. Eriksson S. Biochemistry. 1988; 27: 4258-4265Crossref PubMed Scopus (98) Google Scholar). However, a kinase with the ability to phosphorylate all four deoxynucleoside kinase has to our knowledge not been found previously. From Lactobacillus acidophilus R-26, two heterodimeric deoxynucleoside kinases, a dGK/dAK and a dCK/dAK have been isolated (23Ikeda S. Ma G.T. Ives D.H. Biochemistry. 1994; 33: 5328-5334Crossref PubMed Scopus (10) Google Scholar). The two kinases showed different binding affinities, as dCK/dAK bound to dCTP-Sepharose, whereas dGK/dAK bound to dATP-Sepharose. in Drosophila, a similar arrangement of closely related monomeric proteins with the same size, each specific for one or two of the substrates, seems unlikely, since all deoxynucleoside kinase activities of Dm-dNK were bound to dTMP-Sepharose.The Km values for the four deoxynucleoside substrates (Table III) were compared with those reported for TK1 from human lymphocytes (24Munch-Petersen B. Tyrsted G. Cloos L. J. Biol. Chem. 1993; 268: 15621-15625Abstract Full Text PDF PubMed Google Scholar), TK2 (14Munch-Petersen B. Cloos L. Tyrsted G. Eriksson S. J. Biol. Chem. 1991; 266: 9032-9038Abstract Full Text PDF PubMed Google Scholar), and dCK (15Bohman C. Eriksson S. Biochemistry. 1988; 27: 4258-4265Crossref PubMed Scopus (98) Google Scholar) from human leukemic spleen, and dGK from human brain (16Wang L. Karlsson A. Arnér E.S.J. Eriksson S. J. Biol. Chem. 1993; 268: 22847-22852Abstract Full Text PDF PubMed Google Scholar). With dCyd and dThd, theDm-dNK Km values were in the low micromolar range around 1 μm, and comparable toKm for dCyd found for human dCK (1 μm) and for dThd found for the high affinity form of human TK1 (0.5 μm). The Dm-dNK Km values for dAdo and dGuo were about 100- and 650-fold higher than those for dCyd and dThd, respectively. However, the Dm-dNKKm value for dAdo was in the same range as that found for the human dCK, and the Dm-dNKKm value for dGuo was about 5.5-fold higher than the one found with the human dCK, and 86-fold higher than the one found with the bovine dGK. The kinetics was clearly classical Michaelis-Menten, in contrast to the kinetics of mammalian TKs and dCK, which exhibited cooperative reaction mechanism with their deoxynucleoside substrates (14Munch-Petersen B. Cloos L. Tyrsted G. Eriksson S. J. Biol. Chem. 1991; 266: 9032-9038Abstract Full Text PDF PubMed Google Scholar, 15Bohman C. Eriksson S. Biochemistry. 1988; 27: 4258-4265Crossref PubMed Scopus (98) Google Scholar, 24Munch-Petersen B. Tyrsted G. Cloos L. J. Biol. Chem. 1993; 268: 15621-15625Abstract Full Text PDF PubMed Google Scholar)The specific activities were high for all four Dm-dNK activities, about 30,000 units/mg for dCyd and dThd, and about 36,000 for dAdo and dGuo, corresponding to kcat values of 15 s−1 for the TK and dCK activities, and 18–19 s−1 for the dAK and dGK activities. When compared with thekcat values calculated for the mammalian enzymes, using the subunit masses, those of Dm-dNK were manyfold higher, about 4-fold higher compared with TK1 (14Munch-Petersen B. Cloos L. Tyrsted G. Eriksson S. J. Biol. Chem. 1991; 266: 9032-9038Abstract Full Text PDF PubMed Google Scholar), 200-, 60-, and 45-fold higher compared with the dCK, dAK, and dGK activities of human dCK (15Bohman C. Eriksson S. Biochemistry. 1988; 27: 4258-4265Crossref PubMed Scopus (98) Google Scholar), respectively, and 13,000- and 9000-fold higher compared with the dGK and dAK activities of bovine dGK (16Wang L. Karlsson A. Arnér E.S.J. Eriksson S. J. Biol. Chem. 1993; 268: 22847-22852Abstract Full Text PDF PubMed Google Scholar), respectively. Likewise, the specificity constantskcat/Km for theDm-dNK ranged from 2.6 × 104s−1m−1 to 1.6 × 107 s−1m−1 (TableIII), and were manyfold higher as compared with the corresponding mammalian activities. The lowest specificity constant of Dm-dNK was that for dGuo, but it was an order of magnitude higher than that for human dCK-dGuo (2.7 × 103s−1m−1) and bovine dGK-dGuo (2.8 × 103 s−1m−1). Regarding these relations and the levels of specific activities, it is likely that the dAK and dGK activities of the Dm-dNK play a significant role for the supply of purine deoxynucleotides for replication and repair of DNA in Drosophila cells.The substrate inhibition studies indicated that the four substrates competed with each other (Table IV). dThd was a strong inhibitor of the phosphorylation of dCyd, dAdo, and dGuo with Ki values in the range of Km for dThd. dAdo and dGuo were able to compete with each other with Ki values in the range of their Km values. Furthermore, dAdo and dGuo were relatively efficient inhibitors of the phosphorylation of thymidine. Together, these data indicate that the four substrates are phosphorylated at the same site of the enzyme.The absence of a TK equivalent to the mammalian S-phase specific TK1 was unexpected, as other DNA replication associated enzymes previously have been isolated from Drosophila embryonic cells (25Wernette C.M. Kaguni L.S. J. Biol. Chem. 1986; 261: 14764-14770Abstract Full Text PDF PubMed Google Scholar, 26Kaguni L.S. Rossignol J.-M. Conaway R.C. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2221-2225Crossref PubMed Scopus (71) Google Scholar). Additionally, the absence of a dCK equivalent to the mammalian dCK should be noted. On the other hand, as discussed above, the dCK and TK activities of the Dm-dNK are even more efficient in their phosphorylation capacities than the mammalian kinases.The specificity toward a number of nucleoside substrates showed interesting properties of the Dm-dNK (Table II). Thus, in addition to the four deoxynucleosides, dUrd and FdUrd were phosphorylated to the same degree as dThd and dCyd and, unexpectedly, both arabinosyl furanosyl pyrimidines were phosphorylated. Such a diverse substrate specificity has to our knowledge not been found for other eucaryotic and bacterial deoxynucleoside kinases.Dm-dNK was eluted from the DEAE matrix at essentially the same KCl concentration as human TK2 (14Munch-Petersen B. Cloos L. Tyrsted G. Eriksson S. J. Biol. Chem. 1991; 266: 9032-9038Abstract Full Text PDF PubMed Google Scholar), but also in other aspects there were similarities. Like TK2 (14Munch-Petersen B. Cloos L. Tyrsted G. Eriksson S. J. Biol. Chem. 1991; 266: 9032-9038Abstract Full Text PDF PubMed Google Scholar), Dm-dNK was a monomer of about 30 kDa with dCK activity, showed negligible capacity to phosphorylate AZT, was able to phosphorylate AraT (Table II), and could use CTP as a phosphate donor. However, mammalian TK2 is unable to phosphorylate dAdo or dGuo.With respect to the capacity to phosphorylate the other arabinofuranosyl nucleoside analog, AraC, the Dm-dNK resembles human dCK that had a kcat value of 7 × 10−2 s−1 and a specificity constant of 3.5 × 103 s−1m−1 (15Bohman C. Eriksson S. Biochemistry. 1988; 27: 4258-4265Crossref PubMed Scopus (98) Google Scholar). The Dm-dNK, however, was even more efficient with a 390-fold higher kcatvalue (27 s−1) and a 280-fold higher specificity constant (Table III).From the properties regarding the native and subunit size, substrate specificity, kinetic constants, and substrate interaction, it is rational to propose that the multifunctional efficient deoxynucleoside kinase from Drosophila S-2 cells is an enzyme with unique properties not hitherto found in any organism. Whether the multifunctional Drosophila deoxynucleoside kinase is present in other insects is currently under investigation. The biosynthesis of deoxynucleoside triphosphates in most living organisms is performed by two pathways, the de novo and the salvage pathway. The main enzyme of the de novo pathway is ribonucleotide reductase, which catalyzes the reduction of the 2′-OH group of the nucleoside diphosphates. The enzyme has been extensively studied as to the structure and the complex feedback regulation by the end products, the four deoxynucleoside triphosphates (1Reichard P. Annu. Rev. Biochem. 1988; 57: 349-374Crossref PubMed Scopus (625) Google Scholar, 2Reichard P. Ehrenberg A. Science. 1983; 221: 514-519Crossref PubMed Scopus (383) Google Scholar). An alternative pathway for supplying deoxynucleoside triphosphates for replication and repair of DNA is the salvage pathway. The principal salvage enzymes are the deoxynucleoside kinases, which phosphorylate deoxynucleosides to the corresponding deoxynucleoside monophosphates. In mammalian cells, there are four deoxynucleoside kinases with different cellular localization and specificities, thymidine kinase 1 (TK1), 1The abbreviations used are: TK1, cytoplasmic specific thymidine kinase; TK2, mitochondrial thymidine kinase; AraC, 1-β-d-arabinofuranosylcytosine; AraT, 1-β-d-arabinofuranosylthymine; AZT, 3′-azido-2′,3′-dideoxythymidine; CHAPS, 3-((3-cholamidopropyl)dimethylammonio)-1-propylsulfonic acid; dAdo, deoxyadenosine; dAK, deoxyadenosine kinase; dCK, deoxycytidine kinase; dCyd, deoxycytidine; dGK, deoxyguanosine kinase; dGuo, deoxyguanosine; Dm-dNK, D. melanogasterdeoxynucleoside kinase; dThd, thymidine; dUrd, deoxyuridine; FddThd, 3′-fluoro-2′,3′-dideoxythymidine; FdUrd, 5-fluorodeoxyuridine; TK, thymidine kinase; CV, coefficient of variation.1The abbreviations used are: TK1, cytoplasmic specific thymidine kinase; TK2, mitochondrial thymidine kinase; AraC, 1-β-d-arabinofuranosylcytosine; AraT, 1-β-d-arabinofuranosylthymine; AZT, 3′-azido-2′,3′-dideoxythymidine; CHAPS, 3-((3-cholamidopropyl)dimethylammonio)-1-propylsulfonic acid; dAdo, deoxyadenosine; dAK, deoxyadenosine kinase; dCK, deoxycytidine kinase; dCyd, deoxycytidine; dGK, deoxyguanosine kinase; dGuo, deoxyguanosine; Dm-dNK, D. melanogasterdeoxynucleoside kinase; dThd, thymidine; dUrd, deoxyuridine; FddThd, 3′-fluoro-2′,3′-dideoxythymidine; FdUrd, 5-fluorodeoxyuridine; TK, thymidine kinase; CV, coefficient of variation. thymidine kinase 2 (TK2), deoxycytidine kinase (dCK), and deoxyguanosine kinase (dGK). TK1 is cytosolic and is expressed only in S-phase cells (3Sherley J.L. Kelly T.J. J. Biol. Chem. 1988; 263: 8350-8358Abstract Full Text PDF PubMed Google Scholar, 4Kauffman M.G. Kel" @default.
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• W2079723980 title "Four Deoxynucleoside Kinase Activities from Drosophila melanogaster Are Contained within a Single Monomeric Enzyme, a New Multifunctional Deoxynucleoside Kinase" @default.
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