Purification and Characterization of Wild-Type and Mutant Tk1 Type Kinases From Caenorhabditis Elegans

Caenorhabditis elegans has a single deoxynucleoside kinase-like gene. The sequence is similar to that of human TK1, but besides accepting thymidine as a substrate, the C. elegans TK1 (CeTK1) also phosphorylates deoxyguanosine. In contrast to human TK1, the CeTK1 exclusively exists as a dimer with a molecular mass of ～60 kDa, even if incubated with ATP. Incubation with ATP induces a transition into a more active enzyme with a higher k_cat but unchanged K_m. This activation only occurs at an enzyme concentration in the incubation buffer of 0.5 μg/ml (8.42 nM) or higher. C-terminal deletion of the enzyme results in lower catalytic efficiency and stability.     

Comparative active-site mutation study of human and Caenorhabditis elegans thymidine kinase 1

The first step for the intracellular retention of several anticancer or antiviral nucleoside analogues is the addition of a phosphate group catalysed by a deoxyribonucleoside kinase such as thymidine kinase 1 (TK1). Recently, human TK1 (HuTK1) has been crystallized and characterized using different ligands. To improve our understanding of TK1 substrate specificity, we performed a detailed, mutation-based comparative structure-function study of the active sites of two thymidine kinases: HuTK1 and Caenorhabditis elegans TK1 (CeTK1). Specifically, mutations were introduced into the hydrophobic pocket surrounding the substrate base. In CeTK1, some of these mutations led to increased activity with deoxycytidine and deoxyguanosine, two unusual substrates for TK1-like kinases. In HuTK1, mutation of T163 to S resulted in a kinase with a 140-fold lower K(m) for the antiviral nucleoside analogue 3'-azido-3'-deoxythymidine (AZT) compared with the natural substrate thymidine. The crystal structure of the T163S-mutated HuTK1 reveals a less ordered conformation of the ligand thymidine triphosphate compared with the wild-type structure but the cause of the changed specificity towards AZT is not obvious. Based on its highly increased AZT activity relative to thymidine activity this TK1 mutant could be suitable for suicide gene therapy.     

Valine, not methionine, is amino acid 106 in human cytosolic thymidine kinase (TK1). Impact on oligomerization, stability, and kinetic properties

Cytosolic thymidine kinase (TK1) cDNA from human lymphocytes was cloned, expressed in Escherichia coli, purified, and characterized with respect to the ATP effect on thymidine affinity and oligomerization. Sequence analysis of this lymphocyte TK1 cDNA and 21 other cDNAs or genomic TK1 DNAs from healthy cells or leukemic or transformed cell lines revealed a valine at amino acid position 106. The TK1 sequence in NCBI GenBank(TM) has methionine at this position. The recombinant lymphocyte TK1(Val-106) (rLy-TK1(Val-106)) has the same enzymatic and oligomerization properties as endogenous human lymphocyte TK1 (Ly-TK1); ATP exposure induces an enzyme concentration-dependent reversible transition from a dimer to a tetramer with 20-30-fold higher thymidine affinity (K(m) about 15 and 0.5 microm, respectively). Substitution of Val-106 with methionine to give rLy-TK1(Met-106) results in a permanent tetramer with the high thymidine affinity (K(m) about 0.5 microm), even without ATP exposure. Furthermore, rLy-TK1(Met-106) is considerably less stable than rLy-TK1(Val-106) (t(12) at 15 degrees C is 41 and 392 min, respectively). Because valine with high probability is the naturally occurring amino acid at position 106 in human TK1 and because this position has high impact on the enzyme properties, the Val-106 form should be used in future investigations of recombinant human TK1.     

Characterization of oligomeric and kinetic properties of tomato thymidine kinase 1

The gene encoding thymidine kinase 1 from tomato (toTK1) has in combination with azidothymidine (AZT) recently been proposed as a powerful suicide gene for anticancer gene therapy. The toTK1/AZT combination has been demonstrated to have several advantages for the treatment of glioblastomas because AZT can easily penetrate the blood-brain barrier and toTK1 can efficiently phosphorylate AZT and also AZT-monophosphate. In a pursuit to further understand the properties of toTK1, we examined the oligomerization properties of recombinant toTK1 and its effect on enzyme kinetics. Previously, it has been shown that human TK1 is a dimer in the absence of ATP and a tetramer if preincubated with ATP. However, we show here that ATP preincubation did not result in a structural shift from dimer to tetramer in toTK1. For human TK1 pretreated with ATP, the K(m) value decreased 20-fold, but toTK1's K(m) value did not show a dependence on the presence or absence of ATP. Furthermore, toTK1 was always found in a highly active form.     

Characterization of three essential residues in the conserved ATP-binding region of Epstein-Barr virus thymidine kinase

The thymidine kinase encoded by Epstein-Barr virus (EBV TK) is an important target for antiviral therapy and the treatment of EBV-associated malignancies. Through computer-assisted alignment with other human herpesviral TK proteins, EBV TK was shown to contain a conserved ATP-binding motif as for the other TK enzymes. To investigate functional roles of three highly conserved residues (G294, K297, T298) within this region, site-directed mutagenesis was employed to generate various mutants. The TK enzyme activity and ATP-binding ability of these mutant TK enzymes were determined and compared with EBV wild-type TK (wtTK). Mutant G294V lost its ATP-binding ability and was inactive in enzyme activity assay. As the enzyme activity of G294A was reduced to 20% of that of wtTK, the K(m) for ATP binding of G294A was 48.7 microM as compared with 30.0 microM of EBV wtTK. These results suggested that G294 participates in ATP binding and contributes to maintenance of structure. EBV TK mutants K297E, K297Q, and K297R lost their ATP-binding ability and enzyme activity. However, K297R was shown to have a preference for usage of GTP (K(m): 43.0 microM) instead of ATP (K(m): 87.6 microM) as the phosphate donor. This implies that, in addition to nucleotide binding, K297 was involved in the selection of phosphate donor. While EBV TK mutant T298S retained approximately 80% of wtTK enzyme activity, T298A lost its enzyme activity, suggesting that a hydroxyl group at this position is important for the enzyme activity. Interestingly, T298A retained its ATP-binding ability, suggesting a role of T298 in the catalytic process but not in the coordination of ATP. This study demonstrated that amino acid residues G294, K297, and T298 in the ATP-binding motif of EBV TK enzyme are essential for the enzymatic activity but are involved in different aspects of its action.     

Differences in the kinetic properties of thymidine kinase isoenzymes in unstimulated and phytohemagglutinin-stimulated human lymphocytes

Summary The two thymidine kinases, TK 1 and TK 2, found in phytohemagglutinin-stimulated human lymphocytes and the thymidine kinase, TK 2N, found in unstimulated human lymphocytes were purified and characterized. All three kinases had molecular weights between 70000 and 75000 which increased to 170000–200000 in the presence of 2 mM ATP.Studies on the kinetic properties of the enzymes with thymidine and ATP as the substrates and dTTP as the inhibitor showed clear differences between TK 1 and TK 2, but a close similarity between TK 2 and TK 2N. With thymidine as the variable substrate, TK 1 showed Michaelis-Menten kinetics, whereas TK 2 and TK 2N showed characteristic biphasic kinetics. With ATP as the variable substrate, all three enzymes showed positive cooperative kinetics, but TK 2 and TK 2N lost the cooperativity in the presence of dTTP. The results from inhibition studies showed, that dTTP was a cooperative inhibitor of TK 1 but a non-cooperative inhibitor of TK 2 and TK 2N.     

Kinetic properties of mutant human thymidine kinase 2 suggest a mechanism for mitochondrial DNA depletion myopathy

Thymidine kinase 2 (TK2) is a mitochondrial (mt) pyrimidine deoxynucleoside salvage enzyme involved in mtDNA precursor synthesis. The full-length human TK2 cDNA was cloned and sequenced. A discrepancy at amino acid 37 within the mt leader sequence in the DNA compared with the determined peptide sequence was found. Two mutations in the human TK2 gene, His-121 to Asn and Ile-212 to Asn, were recently described in patients with severe mtDNA depletion myopathy (Saada, A., Shaag, A., Mandel, H., Nevo, Y., Eriksson, S., and Elpeleg, O. (2001) Nat. Genet. 29, 342-344). The same mutations in TK2 were introduced, and the mutant enzymes, prepared in recombinant form, were shown to have similar subunit structure to wild type TK2. The I212N mutant showed less than 1% activity as compared with wild type TK2 with all deoxynucleosides. The H121N mutant enzyme had normal K(m) values for thymidine (dThd) and deoxycytidine (dCyd), 6 and 11 microm, respectively, but 2- and 3-fold lower V(max) values as compared with wild type TK2 and markedly increased K(m) values for ATP, leading to decreased enzyme efficiency. Competition experiments revealed that dCyd and dThd interacted differently with the H121N mutant as compared with the wild type enzyme. The consequences of the two point mutations of TK2 and the role of TK2 in mt disorders are discussed.     

Striking ability of adenosine-2'(3')-deoxy-3'(2')-triphosphates and related analogues to replace ATP as phosphate donor for all four human,and the Drosophila melanogaster,deoxyribonucleoside kinases

In extension of an earlier report, six non-conventional analogues of ATP, three adenosine-2'-triphosphates (3'-deoxy, 3'-deoxy-3'-fluoro- and 3'-deoxy-3'-fluoroxylo-), and three adenosine-3'-triphosphates (2'-deoxy-, 2'-deoxy-2'-fluoro- and 2'-deoxy-2'-fluoroara-), were compared with ATP as potential phosphate donors for human deoxycytidine kinase (dCK), cytosolic thymidine kinase (TK1), mitochondrial TK2, deoxyguanosine kinase (dGK), and the deoxyribonucleoside kinase (dNK) from Drosophila melanogaster. With one group of enzymes, comprising TK1, TK2, dNK and dCK (with dAdo as acceptor), only 3'-deoxyadenosine-2'-triphosphate was an effective donor (5-60% that for ATP), and the other five analogues much less so, or inactive. With a second set, including dCK (dCyd, but not dAdo, as acceptor) and dGK (dGuo as acceptor), known to share high sequence similarity (approximately 45% sequence identity), all six analogues were good to excellent donors (13-119% that for ATP). With dCK and ATP1, products were shown to be 5'-phosphates. With dCK, donor properties of the analogues were dependent on the nature of the acceptor, as with natural 5'-triphosphate donors. With dCK (dCyd as acceptor), Km and Vmax for the two 2'(3')-deoxyadenosine-3'(2')-triphosphates are similar to those for ATP. With dGK, Km values are higher than for ATP, while Vmax values are comparable. Kinetic studies further demonstrated Michaelis-Menten (non-cooperative) or cooperative kinetics, dependent on the enzyme employed and the nature of the donor. The physiological significance, if any, of the foregoing remains to be elucidated. The overall results are, on the other hand, highly relevant to studies on the modes of interaction of nucleoside kinases with donors and acceptors; and, in particular, to interpretations of the recently reported crystal structures of dGK with bound ATP, of dNK with bound dCyd, and associated modeling studies.     

Compulsory order of substrate binding to herpes simplex virus type 1 thymidine kinase. A calorimetric study

Isothermal titration calorimetry has been used to investigate the thermodynamic parameters of the binding of thymidine (dT) and ATP to herpes simplex virus type 1 thymidine kinase (HSV1 TK). Binding follows a sequential pathway in which dT binds first and ATP second. The free enzyme does not bind ATP, whose binding site becomes only accessible in the HSV1 TK.dT complex. At pH 7.5 and 25 degrees C, the binding constants are 1.9 x 10(5) m(-1) for dT and 3.9 x 10(6) m(-1) for ATP binding to the binary HSV1 TK.dT complex. Binding of both substrates is enthalpy-driven and opposed by a large negative entropy change. The heat capacity change (DeltaCp) obtained from DeltaH in the range of 10-25 degrees C is -360 cal K(-1) mol(-1) for dT binding and -140 cal K(-1) mol(-1) for ATP binding. These large DeltaCp values are incompatible with a rigid body binding model in which the dT and ATP binding sites pre-exist in the free enzyme. Values of DeltaCp and TDeltaS strongly indicate large scale conformational adaptation of the active site in sequential substrate binding. The conformational changes seem to be more pronounced in dT binding than in the subsequent ATP binding. Considering the crystal structure of the ternary HSV1 TK.dT.ATP complex, a large movement in the dT binding domain and a smaller but substantial movement in the LID domain are proposed to take place when the enzyme changes from the substrate-free, presumably more open and less ordered conformation to the closed and compact conformation of the ternary enzyme-substrate complex.     

Induction of thymidine kinase and DNase in varicella-zoster virus-infected cells and kinetic properties of the virus-induced thymidine kinase.

Thymidine kinase (TK), DNA polymerase, and DNase activities were induced in human foreskin fibroblasts after varicella-zoster virus infection. The induced TK and DNase activities have electrophoretic mobilities different from the corresponding host enzymes. Varicella-zoster virus-induced TK was purified and separated from the host enzyme by affinity column chromatography. This enzyme has been shown to have a broader substrate specificity with respect to either the phosphate donor or acceptor as compared with human cytoplasmic and mitochondrial TKs. The best phosphate donor is ATP, with a Km of 16 microM. The Km values of thymidine, deoxycytidine, and 5-propyl deoxyuridine were estimated to be 0.4, 180, and 0.8 microM, respectively. The Ki values for several analogs of thymidine such as 5-iododeoxyuridine, arabinofuranosylthymine, 5-ethyl deoxyuridine, and 5-cyanodeoxyuridine were also examined. TTP acted as a noncompetitive inhibitor with respect to thymidine with a Ki of 5 microM. The kinetic behavior of varicella-zoster virus-induced TK is different from human cytoplasmic, human mitochondrial, and herpes simplex virus type 1- and 2-induced TKs.     

Thymidine and 3'-azido-3'-deoxythymidine metabolism in human peripheral blood lymphocytes and monocyte-derived macrophages. A study of both anabolic and catabolic pathways.

3'-Azido-3'-deoxythymidine (AZT) is HIV-inhibitory in human macrophages, which is surprising in view of the low AZT phosphorylation reported in macrophage extracts. To elucidate the mechanism of AZT activation, we studied AZT anabolism as well as catabolism in human lymphocytes and macrophages, and compared it to that of thymidine. Thymidine kinase (TK)-specific activity in mitogen-stimulated lymphocytes was 15 times higher than in macrophages. However, the TK activity per cell was only 1.3 times higher, because of the large macrophage cell volume. Total cellular TK activity, but not specific activity, matched the level of intracellular AZT anabolism. The substrate specificity of TK in macrophages strongly suggests that mitochondrial TK2 was the enzyme phosphorylating thymidine and AZT in these cells, whereas it was cytosolic TK1 in stimulated lymphocytes. In vivo thymidine catabolism was extensive, forming thymine and dihydrothymine. In macrophages more than 95% of the added thymidine (0.5 microM) was degraded within 60 min. AZT, in contrast, was not catabolized, which explains the high AZT nucleotide accumulation, a process opposed only by AZTMP excretion. The lack of catabolism together with phosphorylation by TK2 clarifies how AZT can inhibit human immunodeficiency virus in macrophages. The fact that TK2 and not TK1 phosphorylates AZT in macrophages should have important implications for combination chemotherapy.     

Identification of the ATP-binding domain of vaccinia virus thymidine kinase

Although small in size (20 kDa), the vaccinia virus (VV) thymidine kinase protein (EC 2.7.1.21 TK) is a relatively complex enzyme which must contain domains involved in binding both substrates (ATP and thymidine) and a feedback inhibitor (dTTP), as well as sequences directing the association of individual protein monomers into a functional tetrameric enzyme. Alignment of predicted amino acid sequences of the thymidine kinase genes from a variety of sources was used to identify highly conserved regions as a first step toward locating potential regions housing essential domains. A conserved domain (domain I) near the amino terminus of VV TK protein had characteristics consistent with a nucleotide-binding site. Analysis of the nucleotide substrate specificity of VV TK indicated that ATP acts as the major phosphate donor for thymidine phosphorylation while GTP, CTP, and UTP were inefficient substrates. Site-directed mutagenesis was performed on domain I to generate 11 mutant enzymes. Comparison of the wild-type and mutant proteins with regard to enzyme activity revealed that two of the mutant enzymes, T18 and S19, exhibited enhanced enzyme activity (3.73-fold and 1.35-fold, respectively) relative to the control. The other mutations introduced led to greatly reduced levels of enzyme activity which correlated with a reduced or altered ability of the mutant enzymes to bind ATP as determined by ATP-agarose affinity chromatography. Wild-type VV TK bound to an ATP affinity column could also be eluted with dTTP. Glycerol gradient separation of wild-type TK in the presence or absence of dTTP indicated that dissociation of the tetrameric complex was not the means by which enzymatic inhibition was achieved. Taken together, these results suggest that (i) domain I (amino acids 11-22) of the VV TK corresponds to the ATP-binding site, and (ii) that dTTP is able to interfere with ATP binding, either directly or indirectly, and thereby inhibit enzymatic activity without dissociating the native enzyme.     

Highly purified recombinant varicella Zoster virus thymidine kinase is a homodimer

Recombinant varicella zoster virus (VZV) thymidine kinase (TK) was isolated in a fast and gentle two-step procedure from Escherichia coli. The TK was expressed as a PreScission-cleavable fusion protein and purified by glutathione and ATP affinity chromatography, yielding homogeneous, highly pure VZV TK. The purified enzyme displays enzymatic activities with K(m) values of 0.3 +/- 0.06 microM for the natural substrate thymidine and 11.6 +/- 3.2 microM for ATP, indicating the biochemical equivalence with the viral VZV TK expressed in infected cells. Determinations of the native molecular weight by size exclusion chromatography and native polyacrylamide gel electrophoresis revealed that the pure enzyme is biologically active as a homodimer.     

Mammalian thymidine kinase 2. Direct photoaffinity labeling with [32P]dTTP of the enzyme from spleen, liver, heart and brain.

Thymidine kinase 2 (TK2), also called mitochondrial thymidine kinase, is a pyrimidine deoxyribonucleoside kinase expressed in all cells and tissues. It was recently purified to apparent homogeneity from human leukemic spleen and the active enzyme was shown to be a monomer of a 29-kDa polypeptide. The enzyme is feedback-inhibited by both end products, dCTP and dTTP. Here we show that TK2 purified from several different sources, including purified beef heart mitochondria, could be directly photoaffinity labeled with radioactive dTTP (approximately 18% of all TK2 molecules were cross-linked to dTTP after 20 min of ultraviolet irradiation) or to a lower extent with dCTP. Photo-incorporation was inhibited by the presence of the other effector but also the phosphate donor ATP blocked photolabeling, with dTTP. Addition of nucleoside substrates gave only a marginal inhibition of photo-incorporation. There were no detectable difference in the molecular size of photolabeled TK2 isolated from human spleen, brain or placenta, monkey liver, beef heart and beef heart mitochondria. Nor was there any significant differences in the enzyme kinetic properties of these enzymes. Cleavage of labeled TK2 with cyanogen bromide showed that dTTP was incorporated into a single 3-kDa peptide. TK2 was the only pyrimidine deoxynucleoside kinase expressed in liver, heart and brain. A detailed characterization of the subunit structure and substrate specificity of this enzyme is of importance for the design of new antiviral and cytostatic therapies based on nucleoside analogs.     

An In Vivo C. elegans Model System for Screening EGFR-Inhibiting Anti-Cancer Drugs

The epidermal growth factor receptor (EGFR) is a well-established target for cancer treatment. EGFR tyrosine kinase (TK) inhibitors, such as gefinitib and erlotinib, have been developed as anti-cancer drugs. Although non-small cell lung carcinoma with an activating EGFR mutation, L858R, responds well to gefinitib and erlotinib, tumors with a doubly mutated EGFR, T790M-L858R, acquire resistance to these drugs. The C. elegans EGFR homolog LET-23 and its downstream signaling pathway have been studied extensively to provide insight into regulatory mechanisms conserved from C. elegans to humans. To develop an in vivo screening system for potential cancer drugs targeting specific EGFR mutants, we expressed three LET-23 chimeras in which the TK domain was replaced with either the human wild-type TK domain (LET-23::hEGFR-TK), a TK domain with the L858R mutation (LET-23::hEGFR-TK[L858R]), or a TK domain with the T790M-L858R mutations (LET-23::hEGFR-TK[T790M-L858R]) in C. elegans vulval cells using the let-23 promoter. The wild-type hEGFR-TK chimeric protein rescued the let-23 mutant phenotype, and the activating mutant hEGFR-TK chimeras induced a multivulva (Muv) phenotype in a wild-type C. elegans background. The anti-cancer drugs gefitinib and erlotinib suppressed the Muv phenotype in LET-23::hEGFR-TK[L858R]-expressing transgenic animals, but not in LET-23::hEGFR-TK[T790M-L858R] transgenic animals. As a pilot screen, 8,960 small chemicals were tested for Muv suppression, and AG1478 (an EGFR-TK inhibitor) and U0126 (a MEK inhibitor) were identified as potential inhibitors of EGFR-mediated biological function. In conclusion, transgenic C. elegans expressing chimeric LET-23::hEGFR-TK proteins are a model system that can be used in mutation-specific screens for new anti-cancer drugs.     

Cell cycle regulation and novel structural features of thymidine kinase,an essential enzyme in Trypanosoma brucei

Thymidine kinase (TK) is a key enzyme in the pyrimidine salvage pathway which catalyzes the transfer of the γ‐phosphate of ATP to 2′‐deoxythymidine (dThd) forming thymidine monophosphate (dTMP). Unlike other type II TKs, the Trypanosoma brucei enzyme (TbTK) is a tandem protein with two TK homolog domains of which only the C‐terminal one is active. In this study, we establish that TbTK is essential for parasite viability and cell cycle progression, independently of extracellular pyrimidine concentrations. We show that expression of TbTK is cell cycle regulated and that depletion of TbTK leads to strongly diminished dTTP pools and DNA damage indicating intracellular dThd to be an essential intermediate metabolite for the synthesis of thymine‐derived nucleotides. In addition, we report the X‐ray structure of the catalytically active domain of TbTK in complex with dThd and dTMP at resolutions up to 2.2 Å. In spite of the high conservation of the active site residues, the structures reveal a widened active site cavity near the nucleobase moiety compared to the human enzyme. Our findings strongly support TbTK as a crucial enzyme in dTTP homeostasis and identify structural differences within the active site that could be exploited in the process of rational drug design.     

Inorganic tripolyphosphate (PPP(i)) as a phosphate donor for human deoxyribonucleoside kinases

Inorganic tripolyphosphate (PPP(i)) and pyrophosphate (PP(i)) were examined as potential phosphate donors for human deoxynucleoside kinase (dCK), deoxyguanosine kinase (dGK), cytosolic thymidine kinase (TK1), mitochondrial TK2, and the deoxynucleoside kinase (dNK) from Drosophila melanogaster. PPP(i) proved to be a good phosphate donor for dGK, as well as for dCK with dCyd, but not dAdo, as acceptor substrate, illustrating also the dependence of donor properties on acceptor. Products of phosphorylation were shown to be 5(')-phosphates. In striking contrast to ATP, the phosphorylation reaction follows strict Michaelis-Menten kinetics, with K(m) values of 74 and 92 microM for dCK and dGK, respectively, and V(max) values 40-50% that for ATP. With the other three enzymes, as well as for dCK with dAdo as acceptor, no, or only low levels (相似文献   

Quaternary structure change as a mechanism for the regulation of thymidine kinase 1-like enzymes

The human cytosolic thymidine kinase (TK) and structurally related TKs in prokaryotes play a crucial role in the synthesis and regulation of the cellular thymidine triphosphate pool. We report the crystal structures of the TK homotetramer from Thermotoga maritima in four different states: its apo-form, a binary complex with thymidine, as well as the ternary structures with the two substrates (thymidine/AppNHp) and the reaction products (TMP/ADP). In combination with fluorescence spectroscopy and mutagenesis experiments, our results demonstrate that ATP binding is linked to a substantial reorganization of the enzyme quaternary structure, leading to a transition from a closed, inactive conformation to an open, catalytic state. We hypothesize that these structural changes are relevant to enzyme function in situ as part of the catalytic cycle and serve an important role in regulating enzyme activity by amplifying the effects of feedback inhibitor binding.