Human TK1 Research Articles

Enantiocontrolled Preparation of ϒ-Substituted Cyclohexenones: Synthesis and Kinase Activity Assays of Cyclopropyl-Fused Cyclohexane Nucleosides.

The enantioselective preparation of the two isomers of 4-hydroxy-2-cyclohexanone derivatives 1a,b was achieved, starting from a common cyclohexenone, through asymmetric transfer hydrogenation (ATH) reactions using bifunctional ruthenium catalysts. From these versatile intermediates, a stereoselective route to a cytosine analogue built on a bicyclo [4.1.0]heptane scaffold is described. Nucleoside kinase activity assays with this cyclopropyl-fused cyclohexane nucleoside, together with other related nucleosides (2a–e), were performed, showing that thymine- and guanine- containing compounds have affinity for herpes simplex virus Type 1 (HSV-1) thymidine kinase (TK) but not for human cytosolic TK-1, thus pointing to their selectivity for herpetic TKs but not cellular TKs.

Molecular characterization of equine thymidine kinase 1 and preliminary evaluation of its suitability as a serum biomarker for equine lymphoma

BackgroundThymidine kinase 1 (TK1) plays a key role in the synthesis of deoxythymidine triphosphate (dTTP) and is thus important for DNA replication and cell proliferation. The expression of TK1 is highest during S-phase, and it is rapidly degraded after mitosis. In cancer cells, TK1 is upregulated, resulting in leakage of excess TK1 into the blood. Consequently, serum TK1 has been used as a diagnostic and prognostic cancer biomarker, mainly in human medicine. The aims of this work were to characterize equine TK1 and to evaluate its suitability as a serum biomarker for equine lymphoma.ResultsEquine TK1 was cloned, expressed in E. coli and affinity purified. The purified recombinant horse TK1 showed broad substrate specificity, phosphorylating pyrimidine deoxyribo- and ribonucleosides and, to some extent, purine deoxynucleosides, including anticancer and antiviral nucleoside analogues. ATP was the preferred phosphate donor. Serum TK1 activity was measured in samples collected from horses with confirmed or suspected lymphoma and control horses with and without concurrent diseases. Serum TK1 activity levels were significantly higher in horses with lymphoma (p < 0.0005) and suspected lymphoma (p < 0.02) and in tumour-free groups with diverse diseases (p < 0.03) than in controls without concurrent diseases. There was a significant difference between the lymphoma group and the tumour-free group with diverse diseases (p < 0.0006). Furthermore, receiver operating characteristic analysis revealed a sensitivity of 0.86, a specificity of 0.95 and an AUC (area under the curve) of 0.92 compared to the controls without concurrent diseases, with a sensitivity of 0.97, a specificity of 0.71 and an AUC of 0.88 when compared with the tumour-free group with diverse diseases.ConclusionEquine TK1 showed high specific activity and broader substrate specificity than human TK1. Anticancer and antiviral thymidine analogues were efficiently phosphorylated by horse TK1, suggesting that these analogues might be good candidates for chemotherapy in horses. Serum TK1 activity was significantly higher in horses with lymphoma than in controls. ROC analysis indicated that serum TK1 could serve as a promising cancer biomarker in horses.

Abstract 454: The role of autophagy in multiple myeloma disease progression

Abstract Multiple Myeloma (MM) is an incurable hematologic neoplasm characterized by accumulation of malignant plasma cells in the bone marrow (BM). MM cells establish close ties with the BM, however at late stages they utilize as yet unknown mechanisms to acquire independence from the microenvironment and survive in extramedullary/nutrient deprived sites. Autophagy, a process responsible for degradation of proteins and organelles under stress conditions, has been suggested as a mechanism whereby tumor cells can withstand metabolic stress. Using two newly acquired human myeloma cell lines (HMCL), TK1 and TK2, from the medullary and extramedullary sites of a MM patient respectively, we investigated the ability of autophagy to confer stress/glutamine deprivation tolerance. Results HMCLs require exogenous L-Glutamine (Gln) for sustained proliferation in vitro. However, when TK1 and TK2 were grown under Gln deprivation conditions (8mM, 4mM, 2mM, 0mM) we observed striking differences in their growth response. The TK2 (peripheral blood) cell line continued proliferating until day 14 (p=0.0046), whereas proliferation of the TK1 cell line significantly decreased in the absence of Gln (p=0.0028 between 8mM and 0mM). Furthermore, the proliferative advantage of TK2 was abrogated at 2mM and 0mM of Gln by the use of an autophagy inhibitor (chloroquine [CQ] (p=0.000235 and p=0.000069 respectively), implicating this process in Gln deprivation tolerance of TK2. Additionally, Gln concentration was inversely correlated with TK1 survival, but not TK2, as measured by annexin-V/PI staining. Cell death in TK1 increased at 24h, from 36.022% at Gln 8mM to 56.2% at Gln 0mM, irrespective of autophagy inhibition. In TK2 cells, Gln deprivation did not cause a significant increase in cell death, whereas inhibition of autophagy increased cell death by two-fold. Accordingly, apoptosis (as measured by PARP cleavage) was induced by the addition of CQ only in TK2 under Gln deprivation. Resistance of TK2 cells to metabolic stress also correlated with up-regulation of the autophagic pathway upon CQ exposure, as demonstrated by a higher LC3BII/I turnover and the presence of autophagic vacuoles in TK2 (but not TK1) cells. In addition to upregulation of autophagy, Gln deprivation also induced the expression of glutamine synthetase in TK2 but not TK1 cells. Chemical inhibition of this enzyme by methionine-sulfoximine (5mM) under Gln deprivation conditions was able to induce significant cell death in TK2 and not TK1 (32.57% and 2.53% increase respectively). These data demonstrate that Gln synthesis is an additional mechanism whereby TK2 cells tolerate Gln starvation. In conclusion we have shown that MM survival outside the BM can be attributed to the ability of cells to overcome “glutamine addiction” due to up-regulation of autophagy and ability to synthesise Gln. Thus, autophagy inhibition is an attractive alternative approach to target late-stage, Gln independent, metastatic cells. Citation Format: Ioanna Savvidou, Sahan Chandrasekara, Tiffany Khong, Andrew Spencer. The role of autophagy in multiple myeloma disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 454.

Abstract 1273: A novel molecular target for lung cancer

Abstract The purpose of this study is to provide further evidence that Thymidine Kinase I (TK1) is selectively expressed on the surface of lung cancer cells, and therefore could be utilized as a potential molecular target. TK1 is an enzyme in the pyrimidine salvage pathway whose expression is closely correlated with cell proliferation and cell turnover. It has previously been shown that upregulation of TK1 is an early event in the development of most cancers. Additionally TK1 serum levels in cancer patients correlate with tumor progression and cancer aggressiveness. Moreover TK1 levels in the original tumors have also been shown to correlate directly with tumor recurrence thus making TK1 a useful prognostic marker in clinical oncology. Recent studies in our lab have provided evidence that TK1 is localized on the plasma membrane in lung cancer cells. Using scanning electron microscopy (SEM), flow cytometry, confocal microscopy and tissue staining, we confirmed the presence of TK1 on the cell surface of H460 cells. Using normal lymphocytes as a comparative control we found TK1 expression on lung cancer cells to be significantly increased compared to control cells. For this investigation, we used three custom designed monoclonal antibodies against human TK1 conjugated with FITC, namely CB1, A72 and A74. Using flow cytometry we confirmed the presence of TK1 on the cell surface, our data shows that H460 cells stained positive for TK1 (12% for A72, 19% for A74, and 29% for CB1). Furthermore, confocal microscopy indicated a positive fluorescent signal for A72, A74 and CB1, suggesting the presence of TK1 on the plasma membrane. SEM images of lung cancer cells and normal lymphocytes stained with TK1 antibodies and gold labeling reported a positive gold signal on H460 cells with relatively no signal from human lymphocytes. Immunohistochemistry staining of carcinoma tissue array panels compared with normal tissue array panels indicated a statistical significant difference of expression levels of TK1 between normal lung tissue and lung carcinoma (P value &amp;lt;0.05). Positive staining was considered if more than 5% of the cells were stained. There was negligible staining in normal lung tissue. Using these techniques we confirmed that TK1 is selectively expressed on the surface of H460 cells and not on normal cells. These results suggest that TK1 could be used as a possible molecular target in lung cancer therapy. Further studies are currently ongoing to monitor TK1 expression and progression to help to elucidate the mechanisms behind it's over expression in cancer cells and to track its progression and movement from the cytosol to the cell surface. Citation Format: Edwin J. Velazquez, Evita G. Weagel, Wei Meng, Michelle H. Townsend, Alex Cummonck, Craig Chandler, Michael R. Downey, Richard Robison, Kim L. O’Neill. A novel molecular target for lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1273.

Quaternary structures of recombinant, cellular, and serum forms of Thymidine Kinase 1 from dogs and humans

BackgroundThymidine kinase 1 (TK1) is a salvage enzyme involved in DNA precursor synthesis, and its expression is proliferation dependent. A serum form of TK1 has been used as a biomarker in human medicine for many years and more recently to monitor canine lymphoma. Canine TK1 has not been cloned and studied. Therefore, dog and human TK1 cDNA were cloned and expressed, and the recombinant enzymes characterized. The serum and cellular forms of canine and human TK1 were studied by size-exclusion chromatography and the level of TK1 protein was determined using polyclonal and monoclonal anti-TK1 antibodies.ResultsCanine TK1 phosphorylated the thymidine (dThd) analog 3'-azido-thymidine (AZT) as efficiently as it did dThd, whereas AZT phosphorylation by human TK1 was less efficient than that of dThd. Dog TK1 was also more thermostable and pH tolerant than the human enzyme. Oligomeric forms were observed with both enzymes in addition to the tetrameric and dimeric forms. Cellular TK1 was predominantly seen in dimeric and tetrameric forms, in the case of both dog TK1 from MDCK cells and human TK1 from CEM cells. Active serum TK1 was found mainly in a high molecular weight form, and treatment with a reducing agent shifted the high molecular weight complex to lower molecular weight forms with reduced total activity. Western blot analysis demonstrated a polypeptide of 26 kDa (dog) and 25 kDa (human) for cellular and serum TK1. There was no direct correlation between serum TK1 activity and protein level. It appears that a substantial fraction of serum TK1 is not enzymatically active.ConclusionsThese results suggest that the serum TK1 protein differs from cellular or recombinant forms, is more active in high molecular weight complexes, and is sensitive to reducing agents. The results presented here provide important information for the future development and use of serum TK1 as a diagnostic biomarker in human and veterinary medicine.

Trypanosoma brucei Thymidine Kinase Is Tandem Protein Consisting of Two Homologous Parts, Which Together Enable Efficient Substrate Binding

Trypanosoma brucei causes African sleeping sickness, a disease for which existing chemotherapies are limited by their toxicity or lack of efficacy. We have found that four parasites, including T. brucei, contain genes where two or four thymidine kinase (TK) sequences are fused into a single open reading frame. The T. brucei full-length enzyme as well as its two constituent parts, domain 1 and domain 2, were separately expressed and characterized. Of potential interest for nucleoside analog development, T. brucei TK was less discriminative against purines than human TK1 with the following order of catalytic efficiencies: thymidine > deoxyuridine ≫ deoxyinosine > deoxyguanosine. Proteins from the TK1 family are generally dimers or tetramers, and the quaternary structure is linked to substrate affinity. T. brucei TK was primarily monomeric but can be considered a two-domain pseudodimer. Independent kinetic analysis of the two domains showed that only domain 2 was active. It had a similar turnover number (k(cat)) as the full-length enzyme but could not self-dimerize efficiently and had a 5-fold reduced thymidine/deoxyuridine affinity. Domain 1, which lacks three conserved active site residues, can therefore be considered a covalently attached structural partner that enhances substrate binding to domain 2. A consequence of the non-catalytic role of domain 1 is that its active site residues are released from evolutionary pressure, which can be advantageous for developing new catalytic functions. In addition, nearly identical 89-bp sequences present in both domains suggest that the exchange of genetic material between them can further promote evolution.

Synthesis, Biological Evaluation, and Radioiodination of Halogenated closo-Carboranylthymidine Analogues

The synthesis and initial biological evaluation of 3-carboranylthymidine analogues (3CTAs) that are (radio)halogenated at the closo-carborane cluster are described. Radiohalogenated 3CTAs have the potential to be used in the radiotherapy and imaging of cancer because they may be selectively entrapped in tumor cells through monophosphorylation by human thymidine kinase 1 (hTK1). Two strategies for the synthesis of a (127)I-labeled form of a specific 3CTA, previously designated as N5, are described: (1) direct iodination of N5 with iodine monochloride and aluminum chloride to obtain N5-(127)I and (2) initial monoiodination of o-carborane to 9-iodo-o-carborane followed by its functionalization to N5-(127)I. The former strategy produced N5-(127)I in low yields along with di-, tri-, and tetraiodinated N5 as well as decomposition products, whereas the latter method produced only N5-(127)I in high yields. N5-(127)I was subjected to nucleophilic halogen- and isotope-exchange reactions using Na(79/81)Br and Na(125)I, respectively, in the presence of Herrmann's catalyst to obtain N5-(79/81)Br and N5-(125)I, respectively. Two intermediate products formed using the second strategy, 1-(tert-butyldimethylsilyl)-9-iodo-o-carborane and 1-(tert-butyldimethylsilyl)-12-iodo-o-carborane, were subjected to X-ray diffraction studies to confirm that substitution at a single carbon atom of 9-iodo-o-carborane resulted in the formation of two structural isomers. To the best of our knowledge, this is the first report of halogen- and isotope-exchange reactions of B-halocarboranes that have been conjugated to a complex biomolecule. Human TK1 phosphorylation rates of N5, N5-(127)I, and N5-(79/81)Br ranged from 38.0% to 29.6% relative to that of thymidine, the endogenous hTK1 substrate. The in vitro uptake of N5, N5-(127)I, and N5-(79/81)Br in L929 TK1(+) cells was 2.0, 1.8, and 1.4 times greater than that in L929 TK1(-) cells.

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 Km value decreased 20-fold, but toTK1's Km value did not show a dependence on the presence or absence of ATP. Furthermore, toTK1 was always found in a highly active form.

Abstract 897: Thymidine kinase 1, a novel biomarker specific to the plasma membrane of cancerous cell lines

Abstract Thymidine Kinase 1 (TK1), an important nucleotide salvage pathway enzyme, has been shown to be upregulated in most malignant cancers. Recent studies have shown that TK1 not only has diagnostic and prognostic potential, but could also be used as a molecular target for therapeutic treatments. Previous research from our lab has shown the expression of TK1 on the plasma membrane of lymphoma. This study provides further insight on the role of TK1 as a diagnostic target for several different tumor cell lines. This study analyzed the localization of TK1 in ten different cancer cell lines using flow cytometry (FCM). The lines studied were A375, a malignant melanoma, CAL27, a squamous cell carcinoma derived from tongue, MDA-MB-231, a breast adenocarcinoma, MDA-MB-435S, previously described as a ductal carcinoma, HT29, a colorectal adenocarcinoma, SW620, a colorectal adenocarcinoma, A549, a lung carcinoma, NCI-H460, a large cell lung carcinoma, DU 145, a prostrate carcinoma, and PC3, a prostate adenocarcinoma. FCM was carried out using a polyclonal antibody against human TK1 as the primary antibody and a FITC conjugated secondary antibody. A sample stained with only secondary antibody was used as a negative control for the experiment. Five replicates were analyzed for each cell line. TK1 was found to bind between 48% and 96%, depending on the cell line. Non-cancerous tonsil tissue and normal lymphocytes were used as controls, where TK1 surface binding was less than 20%. These data indicate the feasibility of TK1 as a possible molecular target for immunotherapy in many types of cancer. Further research is ongoing to determine specific immunotherapy treatment options, and to verify the expression of TK1 in other cancer cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 897. doi:10.1158/1538-7445.AM2011-897

Prodrug Activation by Cryptosporidium Thymidine Kinase

Cryptosporidium spp. cause acute gastrointestinal disease that can be fatal for immunocompromised individuals. These protozoan parasites are resistant to conventional antiparasitic chemotherapies and the currently available drugs to treat these infections are largely ineffective. Genomic studies suggest that, unlike other protozoan parasites, Cryptosporidium is incapable of de novo pyrimidine biosynthesis. Curiously, these parasites possess redundant pathways to produce dTMP, one involving thymidine kinase (TK) and the second via thymidylate synthase-dihydrofolate reductase. Here we report the expression and characterization of TK from C. parvum. Unlike other TKs, CpTK is a stable trimer in the presence and absence of substrates and the activator dCTP. Whereas the values of k(cat) = 0.28 s(-1) and K(m)(,ATP) = 140 microm are similar to those of human TK1, the value of K(m)(thymidine) = 48 microm is 100-fold greater, reflecting the abundance of thymidine in the gastrointestinal tract. Surprisingly, the antiparasitic nucleosides AraT, AraC, and IDC are not substrates for CpTK, indicating that Cryptosporidium possesses another deoxynucleoside kinase. Trifluoromethyl thymidine and 5-fluorodeoxyuridine are good substrates for CpTK, and both compounds inhibit parasite growth in an in vitro model of C. parvum infection. Trifluorothymidine is also effective in a mouse model of acute disease. These observations suggest that CpTK-activated pro-drugs may be an effective strategy for treating cryptosporidiosis.

Reversible tetramerization of human TK1 to the high catalytic efficient form is induced by pyrophosphate, in addition to tripolyphosphates, or high enzyme concentration

Thymidine kinase (TK1) is a key enzyme in the salvage pathway of deoxyribonucleotide metabolism, catalyzing the first step in the synthesis of dTTP by transfer of a gamma-phosphate group from a nucleoside triphosphate to the 5'-hydroxyl group of thymidine, forming dTMP. Human TK1 is cytosolic and its activity is absent in resting cells, appears in late G(1), increases in S phase coinciding with the increase in DNA synthesis, and disappears during mitosis. The fluctuation of TK1 through the cell cycle is important in providing a balanced supply of dTTP for DNA replication, and is partly due to regulation of TK1 expression at the transcriptional level. However, TK1 is a regulatory enzyme that can interchange between its dimeric and tetrameric forms, which have low and high catalytic efficiencies, respectively, depending on pre-assay incubation with ATP. Here, the part of ATP that is necessary for tetramerization and how the reaction velocity is influenced by the enzyme concentration are determined. The results show that only two or three of the phosphate groups of ATP are necessary for tetramerization, and that kinetics and tetramerization are closely related. Furthermore, the enzyme concentration was found to have a pivotal effect on catalytic efficiency.

5-Bromovinyl 2′-Deoxyuridine Phosphorylation by Mitochondrial and Cytosolic Thymidine Kinase (TK2 and TK1) and Its Use in Selective Measurement of TK2 Activity in Crude Extracts

Mitochondrial thymidine kinase (TK2) is responsible for phosphorylation of thymidine and deoxycytidine and plays a crucial role in mitochondrial DNA precursor synthesis. TK2 is expressed in all tissues at low levels complicating accurate determinations, especially in tissues with high cytosolic thymidine kinase (TK1) activity. Recently, 5-bromovinyl 2 ′-deoxyuridine (BvdU) at 0.2 μ M was used to measure TK2 activity selectively. BvdU phosphorylation by pure human TK2 and TK1 was tested here, and the ratio of BvdU phosphorylation by TK2/TK1 was 91 at 0.2 μ M but was 500 at 2.5 μ M. Therefore, for reliable measurement of TK2 activity higher BvdU concentration should be used.

Selective Phosphorylation of Antiviral Drugs by Vaccinia Virus Thymidine Kinase

The antiviral activity of a new series of thymidine analogs was determined against vaccinia virus (VV), cowpox virus (CV), herpes simplex virus, and varicella-zoster virus. Several compounds were identified that had good activity against each of the viruses, including a set of novel 5-substituted deoxyuridine analogs. To investigate the possibility that these drugs might be phosphorylated preferentially by the viral thymidine kinase (TK) homologs, the antiviral activities of these compounds were also assessed using TK-deficient strains of some of these viruses. Some of these compounds were shown to be much less effective in the absence of a functional TK gene in CV, which was unexpected given the high degree of amino acid identity between this enzyme and its cellular homolog. This unanticipated result suggested that the CV TK was important in the mechanism of action of these compounds and also that it might phosphorylate a wider variety of substrates than other type II enzymes. To confirm these data, we expressed the VV TK and human TK1 in bacteria and isolated the purified enzymes. Enzymatic assays demonstrated that the viral TK could efficiently phosphorylate many of these compounds, whereas most of the compounds were very poor substrates for the cellular kinase, TK1. Thus, the specific phosphorylation of these compounds by the viral kinase may be sufficient to explain the TK dependence. This unexpected result suggests that selective phosphorylation by the viral kinase may be a promising new approach in the discovery of highly selective inhibitors of orthopoxvirus replication.

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 kcat but unchanged Km. 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.

Transgenic expression of mitochondrial thymidine kinase isoform (TK2) increases mitochondrial DNA (mtDNA) and subsequent myocardial dysfunction following HAART

Two distinct thymidine kinase (TK) isoforms are found in mammalian cells: cytoplasmic (TK1) and mitochondrial (TK2). In addition to the native deoxyribonucleoside substrates, these kinases can phosphorylate a variety of antiviral prodrugs. In antiretroviral therapy TK1 and TK2 play key roles because they catalyze the first step in activation of nucleoside reverse transcriptase inhibitors (NRTIs). NRTIs exhibit mitochondrial toxicity. Two transgenic lines were created that over-expressed the human TK1 or TK2 gene in murine myocardium to decipher mechanisms of toxicity. HAART treatment (including zidovudine, lamivudine and indinavir) were administered by gavage to TG’s and wild type (WT) littermates (35 days) at human doses. Cardiac and mitochondrial structure and function were examined. Left ventricle (LV) mass was defined echocardiographically (ECHO), mitochondrial ultrastructural defects were identified by electron microscopy (EM), and abundance of cardiac mtDNA was quantified by real time PCR. TGs expressing TK1 had no changes in LV mass from WTs, with or without HAART. mtDNA levels also remained constant. In contrast, TGs expressing TK2 with HAART had significant increase in LV mass from WTs with HAART. Cardiac mtDNA significantly increased in TGs expressing TK2 with or without HAART, compared to WTs. Data here suggest that NRTI phosphorylation by TK2 over-expression in the murine heart has a significant effect on mtDNA abundance compared to that of TK1 over-expression. These data suggest that mitochondrial toxicity of NRTIs relates to pharmacological events within mitochondria. Research supported by NIH/NHLBI R01HL072707 and R01HL059798.

High-level expression and purification of human thymidine kinase 1: Quaternary structure, stability, and kinetics

Human cytosolic thymidine kinase (hTK1) is the key enzyme of the pyrimidine salvage pathway and phosphorylates thymidine to thymidine monophosphate, a precursor building block of the DNA. Wild-type hTK1 (hTK1W) as well as a truncated form of the enzyme (hTK1M) carrying deletions at the N- and C-terminal regions were cloned as His 6-tagged fusion proteins. Expression, isolation, and purification protocols have been established, leading to high yields of soluble and active wild type (∼35 mg) and truncated hTK1 (∼23 mg) per liter of culture. The protein was purified to near homogeneity. The chaperone DnaK was identified to be the major contaminant that could be removed by applying an additional ATP–MgCl 2 incubation and washing step. hTK1W was a permanent tetramer in solution, whereas the truncated construct hTK1M appears to be a dimer in absence and presence of substrates. Both hTK1W and hTK1M exhibit pronounced thermal stability with transition temperatures ( T m) of 71.7 and 73.4 °C, respectively, when measured without adding substrates. The presence of substrates stabilized both hTK1W (Δ T m ranging from 5.6 to 12.5 °C) and hTK1M (Δ T m ranging from 0.8 to 5.3 °C). Both enzymes show high activity over a broad range of pH, temperature, and ionic strength. Kinetic studies determined a K M of 0.51 μM and a k cat of 0.28 s −1 for wild-type hTK1. The truncated hTK1M has a K M of 0.87 μM and k cat of 1.65 s −1, thus exhibiting increased catalytic efficiency. The availability of recombinant human TK1 will facilitate further biochemical and crystallographic studies.

Structure of the substrate complex of thymidine kinase from Ureaplasma urealyticum and investigations of possible drug targets for the enzyme

Thymidine kinases have been found in most organisms, from viruses and bacteria to mammals. Ureaplasma urealyticum (parvum), which belongs to the class of cell-wall-lacking Mollicutes, has no de novo synthesis of DNA precursors and therefore has to rely on the salvage pathway. Thus, thymidine kinase (Uu-TK) is the key enzyme in dTTP synthesis. Recently the 3D structure of Uu-TK was determined in a feedback inhibitor complex, demonstrating that a lasso-like loop binds the thymidine moiety of the feedback inhibitor by hydrogen bonding to main-chain atoms. Here the structure with the substrate deoxythymidine is presented. The substrate binds similarly to the deoxythymidine part of the feedback inhibitor, and the lasso-like loop binds the base and deoxyribose moieties as in the complex determined previously. The catalytic base, Glu97, has a different position in the substrate complex from that in the complex with the feedback inhibitor, having moved in closer to the 5'-OH of the substrate to form a hydrogen bond. The phosphorylation of and inhibition by several nucleoside analogues were investigated and are discussed in the light of the substrate binding pocket, in comparison with human TK1. Kinetic differences between Uu-TK and human TK1 were observed that may be explained by structural differences. The tight interaction with the substrate allows minor substitutions at the 3 and 5 positions of the base, only fluorine substitutions at the 2'-Ara position, but larger substitutions at the 3' position of the deoxyribose.

Deoxyribonucleoside kinases: two enzyme families catalyze the same reaction

Mammals have four deoxyribonucleoside kinases, the cytoplasmic (TK1) and mitochondrial (TK2) thymidine kinases, and the deoxycytidine (dCK) and deoxyguanosine (dGK) kinases, which salvage the precursors for nucleic acids synthesis. In addition to the native deoxyribonucleoside substrates, the kinases can phosphorylate and thereby activate a variety of anti-cancer and antiviral prodrugs. Recently, the crystal structure of human TK1 has been solved and has revealed that enzymes with fundamentally different origins and folds catalyze similar, crucial cellular reactions.

Perturbation of ATP-induced tetramerization of human cytosolic thymidine kinase by substitution of serine-13 with aspartic acid at the mitotic phosphorylation site

Human cytosolic thymidine kinase (TK1) is tightly regulated in the cell cycle by multiple mechanisms. Our laboratory has previously shown that in mitotic-arrested cells human TK1 is phosphorylated at serine-13, accompanied by a decrease in catalytic efficiency. In this study we investigated whether serine-13 phosphorylation regulated TK1 activity and found that substitution of serine-13 with aspartic acid (S13D), which mimics phosphorylation, not only diminished the ATP-activating effect on the enzyme, but also decreased its thymidine substrate affinity. Our experimental results further showed that the S13D mutation perturbed ATP-induced tetramerization of TK1. Given that the dimeric form of TK1 is less active than the tetrameric, we propose that mitotic phosphorylation of serine-13 is of physiological importance, in that it may counteract ATP-dependent activation of TK1 by affecting its quaternary structure, thus attenuating its enzymatic function at the G2/M phase.

Novel deoxynucleoside-phosphorylating enzymes in mycoplasmas: evidence for efficient utilization of deoxynucleosides.

Mycoplasmas are unable to synthesize purine and pyrimidine bases de novo. Therefore, salvage of existing nucleosides and bases is essential for their survival. Four mycoplasma species were studied with regard to their ability to phosphorylate deoxynucleosides. High levels of thymidine kinase (TK), deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK) and deoxyadenosine kinase (dAK) activities were detected in extracts from Mycoplasma pneumoniae, Mycoplasma mycoides subsp. mycoides SC (M. mymySC), Acholeplasma laidlawii (A. laidlawii) and Mycoplasma arginini (M. arginini). Nucleoside phosphotransferase activities were found at high levels in A. laidlawii and low levels in M. arginini. Pyrophosphate-dependent deoxynucleoside kinase activities were detected mainly in A. laidlawii and M. mymySC extracts. Two open reading frames were identified in the M. mymySC genome; one showed 25% sequence identity to human dGK and the other one had about 26% sequence identity to human TK1. The M. mymySC dGK-like enzyme was cloned, expressed in Escherichia coli and affinity-purified. This enzyme phosphorylated dAdo, dGuo and dCyd, and the highest catalytic rate was with dAdo as substrate. Therefore, we suggest that this enzyme should be named deoxyadenosine kinase. The physiological role of mycoplasma dAK and TK may be to support the unusually large dATP and dTTP pools required for replication of mycoplasma genomes.