Valine, Not Methionine, Is Amino Acid 106 in Human Cytosolic Thymidine Kinase (TK1)

Berenstein, Dvora; Christensen, J F; Kristensen, Tina; Hofbauer, Roland; Munch‐Petersen, Birgitte

doi:10.1074/jbc.m005325200

Cited by 28 publications

(58 citation statements)

References 25 publications

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“…K m values were confirmed in a standard spectrophotometric assay (50) and an isothermal calorimetry assay that directly measures the heat of binding of the substrate to the enzyme and generated values of 27 and 24 M, respectively (P. F. Torrence and R. F. Smith, unpublished data). These values agree well with the reported value of 15 M for the human TK1 homolog purified from E. coli, which occurs predominantly as a dimer (4). These data suggested that the assay conditions were suitable for assessing the kinetic parameters of other potential substrates.…”

Section: Antiviral Activity and Tk Dependence In Orthopoxvirusessupporting

confidence: 82%

Selective Phosphorylation of Antiviral Drugs by Vaccinia Virus Thymidine Kinase

Prichard¹,

Keith²,

Johnson³

et al. 2007

Antimicrob Agents Chemother

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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.

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Section: Antiviral Activity and Tk Dependence In Orthopoxvirusessupporting

confidence: 82%

Selective Phosphorylation of Antiviral Drugs by Vaccinia Virus Thymidine Kinase

Prichard¹,

Keith²,

Johnson³

et al. 2007

Antimicrob Agents Chemother

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“…Amino acid 106 has been shown to have high impact on the enzymatic and oligomerization properties of TK1, with the valine 106 TK1 form behaving essentially as the endogenous TK1 (25,27). Comprehensive sequencing studies verify that valine is, in fact, the naturally occurring amino acid at site 106 (25,28,29). Expression and purification of TK1 was performed as described in ref.…”

Section: Methodsmentioning

confidence: 99%

“…24. LyTK1Val106 is derived from the tk1 gene from human lymphocytes (25), where codon 106 encodes a valine instead of a methionine as reported for the original tk1 clone (26). Amino acid 106 has been shown to have high impact on the enzymatic and oligomerization properties of TK1, with the valine 106 TK1 form behaving essentially as the endogenous TK1 (25,27).…”

Section: Methodsmentioning

confidence: 99%

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Structures of thymidine kinase 1 of human and mycoplasmic origin

Welin

Kosinska

Mikkelsen

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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117

167

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Cytosolic thymidine kinase 1, TK1, is a well known cell-cycleregulated 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 ␣͞␤-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 ␤-structures at the root of a ␤-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

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“…2,3 In addition, a modification of the sequence for TK1 has been described. 4 TK1, which is located in the cytoplasm, is a cell cycleregulated enzyme. 5 Its activity fluctuates with DNA synthesis, being high in dividing and malignant cells and low in quiescent cells.…”

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confidence: 99%

Thymidine kinase in epithelial ovarian cancer: Relationship with the other pyrimidine pathway enzymes

Fujiwaki

Hata

Nakayama

et al. 2002

Intl Journal of Cancer

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TK is a pyrimidine metabolic pathway enzyme involved in salvage DNA synthesis. What roles TK may play in epithelial ovarian cancer and the relationships between TK and the other pyrimidine pathway enzymes remain unclear. We examined TK1 gene expression by RT-PCR and related it to gene expression of TS, TP and DPD in 69 samples from epithelial ovarian cancer, 8 low-malignant-potential tumors, 16 benign ovarian tumors and 34 normal ovaries. Additionally, cytosolic and serum TK activities were determined by radioenzymatic assay. TK1 gene expression, the ratio of TK1 to TS gene expression, that of TK1 to TP and that of TK1 to DPD were significantly higher in epithelial ovarian cancer than in normal ovaries. In epithelial ovarian cancer, TK1 gene expression correlated with cytosolic and serum TK activities, Key words: thymidine kinase; thymidylate synthase; thymidine phosphorylase; dihydropyrimidine dehydrogenase; epithelial ovarian cancer; radioenzymatic assay; capecitabine TK is an important pyrimidine metabolic pathway enzyme, which catalyzes the phosphorylation of thymidine to deoxythymidine monophosphate using either exogenous or endogenous metabolic thymidine as a substrate. 1 This salvage pathway is the only one by which thymidine is introduced into DNA synthesis. Two isoforms of the TK gene (TK1 and TK2) have been described. 2,3 In addition, a modification of the sequence for TK1 has been described. 4 TK1, which is located in the cytoplasm, is a cell cycleregulated enzyme. 5 Its activity fluctuates with DNA synthesis, being high in dividing and malignant cells and low in quiescent cells. 6,7 TK2 is considered to be located in the mitochondria; levels of TK2 remain relatively constant throughout the cell cycle. Clinical studies have reported elevated serum TK activity in various human malignancies. 8 The majority of these studies concerned hematologic malignancies, but serum TK activity was also elevated in patients with epithelial ovarian cancer. 9 Additionally, serum and cytosolic TK activities appear to have prognostic value in some solid tumors, such as breast, 7,10,11 small cell lung, 12 prostate 13 and cervical 14 cancers. However, little is known about the clinical and prognostic value of tissue TK gene expression in human cancers.TS, TP, DPD and TK are pyrimidine pathway enzymes for de novo DNA synthesis or salvage nucleotide synthesis. TS is an enzyme for the de novo synthesis of deoxythymidine monophosphate (the same product as TK) and is associated with cell proliferative activity. 15,16 TP catalyzes the reversible phosphorolysis of thymidine (the same substrate as TK) to thymine and 2-deoxy-Dribose-1 phosphate 17 and has angiogenic and antiapoptotic effects. 18,19 Subsequently, thymine is degraded to dihydrothymine by DPD, which is inversely related to malignant behavior. 20,21 These 3 enzymes are also responsible for the efficacy and metabolism of fluoropyrimidine cytostatic agents. TS is a critical target enzyme of these drugs. 15 The new agent capecitabine (N 4 -pentyloxycarbonyl-5Ј-deoxy-5-fluo...

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Valine, Not Methionine, Is Amino Acid 106 in Human Cytosolic Thymidine Kinase (TK1)

Cited by 28 publications

References 25 publications

Selective Phosphorylation of Antiviral Drugs by Vaccinia Virus Thymidine Kinase

Selective Phosphorylation of Antiviral Drugs by Vaccinia Virus Thymidine Kinase

Structures of thymidine kinase 1 of human and mycoplasmic origin

Thymidine kinase in epithelial ovarian cancer: Relationship with the other pyrimidine pathway enzymes

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