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Mitochondrial thymidine kinase 2 (TK2) is a nuclear gene-encoded protein, synthesized in the cytosol and subsequently translocated into the mitochondrial matrix, where it catalyzes the phosphorylation of thymidine (dT) and deoxycytidine (dC). The kinetics of dT phosphorylation exhibits negative cooperativity, but dC phosphorylation follows hyperbolic Michaelis-Menten kinetics. The two substrates compete with each other in that dT is a competitive inhibitor of dC phosphorylation, while dC acts as a noncompetitive inhibitor of dT phosphorylation. In addition, TK2 is feedback inhibited by dTTP and dCTP. TK2 also phosphorylates a number of pyrimidine nucleoside analogues used in antiviral and anticancer therapy and thus plays an important role in mitochondrial toxicities caused by nucleoside analogues. Deficiency in TK2 activity due to genetic alterations causes devastating mitochondrial diseases, which are characterized by mitochondrial DNA (mtDNA) depletion or multiple deletions in the affected tissues. Severe TK2 deficiency is associated with early-onset fatal mitochondrial DNA depletion syndrome, while less severe deficiencies result in late-onset phenotypes. In this review, studies of the enzyme kinetic behavior of TK2 enzyme variants are used to explain the mechanism of mtDNA depletion caused by TK2 mutations, thymidine overload due to thymidine phosphorylase deficiency, and mitochondrial toxicity caused by antiviral thymidine analogues.

The pathogenic mycoplasma Ureaplasma parvum (Up) causes opportunistic infections and relies on salvage of nucleosides for DNA synthesis and Up thymidine kinase (UpTK) provides the necessary thymidine nucleotides. The anti-HIV compound 3 -azido-3'-deoxythymidine (AZT) is a good substrate for TK. Methods for a rapid and efficient synthesis of new 3'-alpha-[1,2,3]triazol-3'-deoxythymidine analogs from AZT under Huisgen conditions are described. Thirteen 3'-analogues were tested with human cytosolic thymidine kinase (hTK1) and UpTK. The new analogs showed higher efficiencies (K(m)/V(max) values) in all cases with UpTK than with hTK1. Still, hTK1 was preferentially inhibited by 9 out of 10 tested analogs. Structural models of UpTK and hTK1 were constructed and used to explain the kinetic results. Two different binding modes of the nucleosides within the active sites of both enzymes were suggested with one predominating in the bacterial enzyme and the other in hTK1. These results will aid future development of anti-mycoplasma nucleosides., ((c) 2010 Elsevier Ltd. All rights reserved.)

Cytosolic thymidine kinase 1, TK1, is a well known cell-cycle-regulated enzyme of importance in nucleotide metabolism as well as an activator of antiviral and anticancer drugs such as 3'-azido3'-deoxythymidine (AZT). We have now determined the structures of the TK1 family, the human and Ureaplasma urealyticum enzymes, in complex with the feedback inhibitor dTTP. The TKls have a tetrameric structure in which each subunit contains an [alpha]/[beta]domain that is similar to ATPase domains of members of the RecA structural family and a domain containing a structural zinc. The zinc ion connects [beta]-structures at the root of a [beta]-ribbon that forms a stem that widens to a lasso-type loop. The thymidine of dTTP is hydrogen-bonded to main-chain atoms predominantly coming from the lasso loop. This binding is in contrast to other deoxyribonucleoside kinases where specific interactions occur with side chains. The TK1 structure differs fundamentally from the structures of the other deoxyribonucleoside kinases, indicating a different evolutionary origin. crystal structures | deoxynucleotide metabolism | prodrug activation

Mitochondrial thymidine kinase 2 (TK2) is an essential enzyme for mitochondrial dNTP synthesis in many tissues. Deficiency in TK2 activity causes devastating mitochondrial diseases. Here we investigated several residues involved in substrate binding and catalysis. We showed that mutations of Gln-110 and Glu-133 affected Mg2+ and ATP binding, and thus are crucial for TK2 function. Furthermore, mutations of Gln-110 and Tyr-141 altered the kinetic behavior, suggesting their involvement in substrate binding through conformational changes. Since the 3 D structure of TK2 is still unknown, and thus, the identification of key amino acids for TK2 function may help to explain how TK2 mutations cause mitochondrial diseases. [ABSTRACT FROM AUTHOR]

Background: Thymidine 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. Results: Equine 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. Conclusion: Equine 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 FROM AUTHOR]

Background: Thymidine kinase 1 (TK1) catalyzes the initial phosphorylation of thymidine in the salvage pathway synthesis of dTTP, an essential building block of DNA. TK1 is a cytosolic enzyme with its highest level during the S-phase of the cell cycle. In cancer cells TK1 is upregulated and excess TK1 is leaked into the blood. Therefore, serum TK1 has been used as biomarker for cancer diagnosis and prognosis in human medicine. Feline TK1 shows high sequence similarity to TK1 from other species. The aim of this study was to characterize feline TK1 and evaluate if serum TK1 can be used as a diagnostic biomarker. Results: Feline TK1 was cloned, expressed and affinity purified. The purified feline TK1 phosphorylated not only pyrimidine deoxyribonucleosides but also pyrimidine ribonucleosides and to some extent purine deoxynucleosides. A number of anticancer and antiviral nucleoside analogs also served as substrates with fairly high efficiency. ATP and dATP were the preferred phosphate donor. Serum TK1 activity in felines with malignant diseases was significantly higher than that in healthy individuals. ROC analysis revealed an area under the curve (AUC) of 0.98 with a sensitivity of 0.83 and a specificity of 0.95 for felines with lymphoma. Serum TK1 activity in felines with IBD or inflammatory disease was within the same range as healthy ones. Furthermore, in felines with lymphoma serum TK1 activity returned to normal levels in response to treatment. Conclusion: Feline TK1 has high specific activity and a broader substrate specificity in comparison with TK1 from other species. Serum TK1 activity in felines with malignant diseases is significantly higher than that in normal felines and in felines with inflammatory diseases. These results suggest that serum TK1 may be a promising biomarker for the diagnosis and monitoring of malignant diseases and for the differential diagnosis of certain inflammatory disease. [ABSTRACT FROM AUTHOR]

Thymidine kinase 1 (TK1) and deoxycytidine kinase (dCK) are required for the activation of thymidine and deoxycytidine analogs used in antiviral and anticancer therapies. Many anticancer drugs cause oxidative stress, and the rise of GSSG and other reactive oxygen species may lead to alteration in gene expression, protein, nucleic acids and lipid modifications. Here, we investigated the effects of oxidative stress and nucleoside analog on the expression and activity of TK1 and dCK. Treatment with GSSG resulted in glutathionylation of dCK and dGK but not TK1 and Dm-dNK, and glutathionylation led to increased dCK activity but decreased dGK activity. Treatment with hydrogen peroxide resulted in induction of TK1, however, the TK1 activity did not correlate with TK1 protein levels, indicating that TK1 protein was inactive. The cellular expression of dCK, however, was reduced but dCK activity was not affected at concentration ≤ 4 mM. Treatment with TFT or 5FdU resulted in downregulation of both TK1 and dCK. However, araC and dFdC treatment led to increased dCK protein but decreased dCK activity. In contrast, both TK1 protein and activity were upregulated after araC and dFdC treatment. Doxorubicin treatment led to upregulation of the TK1 but downregulation of dCK. In conclusion TK1 and dCK expression and activity are apparently affected by oxidative stress and treatment by nucleoside analogs. These results demonstrate the pharmacokinetic importance of characterizing the expression and activity of TK1 and dCK during chemotherapy with thymidine and deoxycytidine analogs in order to optimize their efficacy. [ABSTRACT FROM AUTHOR]

Mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) catalyze the initial phosphorylation of pyrimidine and purine deoxyribonucleosides, and are essential for maintaining mitochondrial dNTP pools for mitochondrial DNA replication. Here the expression of mitochondrial TK2 and dGK in relation to cell growth phases in cultured cells was investigated. TK2 and dGK protein levels in isolated mitochondria and TK2 activity in total cell extracts from U2OS and TK1 deficient L929 cells were determined. We found that TK2 levels were negatively correlated with cell growth rates and there was an exponential increase in TK2 levels in cells entering stationary phase. The expression of dGK did not change and appeared to be constitutive. [ABSTRACT FROM AUTHOR]

A library of purine and pyrimidine nucleosides modified with carborane or metallacarborane boron clusters at different locations, consisting of new molecules as well as already described compounds, was prepared. The compounds were tested as substrates for human deoxynucleoside kinases. Some conjugates, with modification attached to N3 of thymidineviaa linker containing the triazole moiety, were efficiently phosphorylated by cytosolic thymidine kinase 1 and mitochondrial thymidine kinase 2. Higher phosphorylation levels were observed with thymidine kinase 1, the phosphorylation of nucleosides modified with metallacarboranes was observed for the first time. [ABSTRACT FROM PUBLISHER]

Background: Thymidine 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. Results: Canine 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. Conclusions: These 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Thymidine kinase (TK) is the key enzyme in salvaging thymidine to produce thymidine monophosphate. Owing to its ability to phosphorylate nucleoside analogue prodrugs, TK has gained attention as a rate-limiting drug activator. We describe the structures of two bacterial TKs, one from the pathogen Bacillus anthracis in complex with the substrate dT, and the second from the food-poison-associated Bacillus cereus in complex with the feedback inhibitor dTTP. Interestingly, in contrast with previous structures of TK in complex with dTTP, in this study dTTP occupies the phosphate donor site and not the phosphate acceptor site. This results in several conformational changes compared with TK structures described previously. One of the differences is the way tetramers are formed. Unlike B. anthracis TK, B. cereus TK shows a loose tetramer. Moreover, the lasso-domain is in open conformation in B. cereus TK without any substrate in the active site, whereas in B. anthracis TK the loop conformation is closed and thymidine occupies the active site. Another conformational difference lies within a region of 20 residues that we refer to as phosphate-binding β-hairpin. The phosphate-binding β-hairpin seems to be a flexible region of the enzyme which becomes ordered upon formation of hydrogen bonds to the α-phosphate of the phosphate donor, dTTP. In addition to descriptions of the different conformations that TK may adopt during the course of reaction, the oligomeric state of the enzyme is investigated. [ABSTRACT FROM AUTHOR]

Bacillus anthracis, which causes anthrax, has attracted attention because of its potential use as a biological weapon. The risk of multidrug resistance against B. anthracis increases the need for antibiotics with new molecular targets. Nucleoside analogs are well-known antiviral and anticancer prodrugs, and thymidine kinase catalyzes the rate-limiting step in the activation of pyrimidine nucleoside analogs used in chemotherapy. The thymidine kinase gene from B. anthracis Sterne strain (34F2) ( Ba-TK) was cloned and expressed in E. coli, and the product was purified and characterized regarding its substrate specificity. Ba-TK phosphorylated pyrimidine nucleosides and all natural nucleoside triphosphates served as phosphate donors. Size exclusion chromatography indicated a dimeric form of Ba-TK, regardless of the presence of ATP. Thymidine was the most efficient substrate with a low Km value (0.6 μM) and a Vmax of 3.3 μmol dTMP mg-1 min-1, but deoxyuridine ( Km=4.2 μM, Vmax=4.1 μmol dUMP mg-1 min-1) was also a good substrate. Several pyrimidine analogs were also tested and analogs with 5-position modifications showed higher activities compared to analogs with 3′- and N3-position modifications. Deoxyuridine analogs were the most potent inhibitors of B. anthracis growth in vitro. These results may be used to guide future development of nucleoside analogs against B. anthracis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cytosolic thymidine kinase 1, TK1, is a well known cell-cycle-regulated enzyme of importance in nucleotide metabolism as well as an activator of antiviral and anticancer drugs such as 3'-azido-3'-deoxythymidine (AZT). We have now determined the structures of the TK1 family, the human and Ureaplasma urealyticum enzymes, in complex with the feedback inhibitor dTTP. The TK1s have a tetrameric structure in which each subunit contains an α/β-domain that is similar to ATPase domains of members of the RecA structural family and a domain containing a structural zinc. The zinc ion connects β-structures at the root of a β-ribbon that forms a stem that widens to a lasso-type loop. The thymidine of dTTP is hydrogen-bonded to main-chain atoms predominantly coming from the lasso loop. This binding is in contrast to other deoxyribonucleoside kinases where specific interactions occur with side chains. The TK1 structure differs fundamentally from the structures of the other deoxyribonucleoside kinases, indicating a different evolutionary origin. [ABSTRACT FROM AUTHOR]

In the present study, we investigated the effect of fluoropyrimidines on the growth of Ureaplasma urealyticum. Addition of fluoropyrimidines strongly inhibited bacterial growth. Growth inhibition by these analogues could be reversed by addition of either thymidine or deoxyuridine, suggesting inhibition of thymidylate biosynthesis as the mechanism in operation. [ABSTRACT FROM AUTHOR]

Mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) catalyze the initial rate limiting phosphorylation of deoxynucleosides and are essential enzymes for mitochondrial function. Chemotherapy using nucleoside analogs is often associated with mitochondrial toxicities. Here we showed that incubation of U2OS cells with didanosine (ddI, 2',3'-dideoxyinosine), a purine nucleoside analog used in the highly active antiretroviral therapy (HAART), led to selective degradation of both mitochondrial TK2 and dGK while the cytosolic deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) were not affected. Addition of guanosine to the ddI-treated cells prevented the degradation of mitochondrial TK2 and dGK. The levels of intracellular reactive oxygen species and protein oxidation in ddI-treated and control cells were also measured. The results suggest that down-regulation of mitochondrial TK2 and dGK may be a mechanism of mitochondrial toxicity caused by antiviral and anticancer nucleoside analogs. [ABSTRACT FROM AUTHOR]