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.     

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 micro 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 micro M but was 500 at 2.5 micro M. Therefore, for reliable measurement of TK2 activity higher BvdU concentration should be used.     

Serum thymidine kinase (TK) evaluation in HIV infection

Serum thymidine kinase (TK), measured using Prolifigen TK-REA, from AB Sangtec Medical, was investigated in 24 HIV seropositive patients without immunological alterations, 26 seropositives with immunological alterations, 125 LAS, 25 ARC, and 20 AIDS. Subjects with serological markers of prior EBV, HBV, and CMV infection were included but none with acute infectious mononucleosis or acute viral hepatitis. Serum TK was elevated from the beginning of the HIV infection, the seropositive stage, and more markedly afterwards during the course of the infection, with a close correlation with the stage. TK also increased during AZT treatment, due to bone-marrow toxicity. On lowering the dosage or discontinuing the drug TK returned to basal levels. Although the rise in serum may well not be correlated only with the HIV infection, it does add to the picture given by other clinical and/or laboratory methods. Serum TK can be a helpful laboratory test in the follow-up of patients with HIV infection, especially when serum levels are disproportionate to the stage, opportunistic infections, lymphoproliferative malignancies. In such cases bone-marrow toxicity due to treatment can be suspected.     

X-irradiation effects on thymidine kinase (TK): I. TK1 and 2 in normal and malignant cells

Abstract. The effect of radiation on TK is more complicated than would be expected from earlier results on bone marrow cells ( Feinendegen et al. 1984 , Int. J. Radiat. Biol. 45, 205). TK activity increased at 0.01 Gy and then decreased up to 1 Gy in mouse spleen. In contrast to the results for the spleen, an increase in activity at 0.1 Gy was seen in mouse thymus. The activity of dephosphorylated TK1 (TK1a) in both spleen and thymus was reduced to 50% after irradiation at 0.5–1 Gy. The degree of phosphorylation (TK1b/TK1a ratio) changed in spleen, but not in thymus. The activity of TK2 in mouse liver increased at 3 h after 5 Gy by about 60%. In mouse ascites tumour, a dose-independent (1–5 Gy) oscillating TK1 activity was found up to 24 h, especially for TK1a and TK1b. The amount of TK1 was unchanged up to 12 h, but decreased at 24 h. This suggests that the differences in the changes in the degree of phosphorylation of TK1 after irradiation among spleen, thymus and ascites tumour further underline the complexity of the response of TK1 activity to irradiation. The dramatic change in the activities of TK1a and TK1b may illustrate that both of them are more radiosensitive than TK-h, a variant with mixed TK1 and TK2 properties.     

Substrate/inhibitor properties of human deoxycytidine kinase (dCK) and thymidine kinases (TK1 and TK2) towards the sugar moiety of nucleosides, including O'-alkyl analogues.

Nucleoside analogues with modified sugar moieties have been examined for their substrate/inhibitor specificities towards highly purified deoxycytidine kinase (dCK) and thymidine kinases (tetrameric high-affinity form of TK1, and TK2) from human leukemic spleen. In particular, the analogues included the mono- and di-O'-methyl derivatives of dC, dU and dA, syntheses of which are described. In general, purine nucleosides with modified sugar rings were feebler substrates than the corresponding cytosine analogues. Sugar-modified analogues of dU were also relatively poor substrates of TK1 and TK2, but were reasonably good inhibitors, with generally lower Ki values vs TK2 than TK1. An excellent discriminator between TK1 and TK2 was 3'-hexanoylamino-2',3'-dideoxythymidine, with a Ki of approximately 600 microM for TK1 and approximately 0.1 microM for TK2. 3'-OMe-dC was a superior inhibitor of dCK to its 5'-O-methyl congener, consistent with possible participation of the oxygen of the (3')-OH or (3')-OMe as proton acceptor in hydrogen bonding with the enzyme. Surprisingly alpha-dT was a good substrate of both TK1 and TK2, with Ki values of 120 and 30 microM for TK1 and TK2, respectively; and a 3'-branched alpha-L-deoxycytidine analogue proved to be as good a substrate as its alpha-D-counterpart. Several 5'-substituted analogues of dC were good non-substrate inhibitors of dCK and, to a lesser extent, of TK2. Finally, some ribonucleosides are substrates of the foregoing enzymes; in particular C is a good substrate of dCK, and 2'-OMe-C is an even better substrate than dC.     

Human thymidine kinase: purification and some properties of the TK1 isoenzyme from placenta

Summary Human thymidine kinase TK1 isoenzyme has been purified 1 800-fold from placenta to a specific activity of 2.9 nmoles/min/mg of protein. The rapid purification procedure includes affinity chromatography on a thymidine-Sepharose column. At all stages of purification, the enzyme showed irreversible lability. The native molecular weight was determined to be 45 000. Human placental TK1 exhibited specificity for ATP and thymidine as substrates, and significant inhibition was found only with thymidine nucleotides. TTP was the most effective inhibitor.     

X-irradiation effects on thymidine kinase (TK): II. The significance of deoxythymidine triphosphate for inhibition of TK1 activity

Abstract. The purpose of this study was to investigate the mechanism behind the high sensitivity of thymidine kinase 1 (TK1) to X-irradiation. The deoxythymidine triphosphate (dTTP) pool was studied in mouse ascites tumour cells 1–24 h after X-irradiation with 5 Gy. Irradiation changed the Michaelis-Menten kinetics of TK1 from linear to biphasic, showing a negative co-operativity. These changes were closely related to changes in the dTTP pool. Addition of dTTP to the cell extract of non-irradiated cells, or thymidine (dTdR) to the culture medium, resulted in changes very similar to the kinetics found in the irradiated cells. Addition of 5¢-amino-5¢-deoxythymidine (5¢-AdTdR), a thymidine analogue that eliminated the inhibitory effect of dTTP on TK1 activity, completely abolished the irradiation-induced inhibition of TK1 activity. We suggest that the reduced TK1 activity is mainly due to an elevated intracellular concentration of dTTP.     

Localization and ordering of acid alpha-glucosidase (GAA) and thymidine kinase (TK1) by fluorescence in situ hybridization

Genomic DNA clones of human acid alpha glucosidase (GAA) and thymidine kinase (TK1) were used to map the exact location and order of these genes on human chromosome 17. Both genes were localized to the 17825-gter band (17825.2–825.3), with GAA distal to TK1. They were also shown to be, respectively, distal and proximal to an anonymous cosmid (cK17.71) previously mapped to this region.     

Mitochondrial thymidine kinase and the enzymatic network regulating thymidine triphosphate pools in cultured human cells

In non-proliferating cells mitochondrial (mt) thymidine kinase (TK2) salvages thymidine derived from the extracellular milieu for the synthesis of mt dTTP. TK2 is a synthetic enzyme in a network of cytosolic and mt proteins with either synthetic or catabolic functions regulating the dTTP pool. In proliferating cultured cells the canonical cytosolic ribonucleotide reductase (R1-R2) is the prominent synthetic enzyme that by de novo synthesis provides most of dTTP for mt DNA replication. In non-proliferating cells p53R2 substitutes for R2. Catabolic enzymes safeguard the size of the dTTP pool: thymidine phosphorylase by degradation of thymidine and deoxyribonucleotidases by degradation of dTMP. Genetic deficiencies in three of the participants in the network, TK2, p53R2, or thymidine phosphorylase, result in severe mt DNA pathologies. Here we demonstrate the interdependence of the different enzymes of the network. We quantify changes in the size and turnover of the dTTP pool after inhibition of TK2 by RNA interference, of p53R2 with hydroxyurea, and of thymidine phosphorylase with 5-bromouracil. In proliferating cells the de novo pathway dominates, supporting large cytosolic and mt dTTP pools, whereas TK2 is dispensable, even in cells lacking the cytosolic thymidine kinase. In non-proliferating cells the small dTTP pools depend on the activities of both R1-p53R2 and TK2. The activity of TK2 is curbed by thymidine phosphorylase, which degrades thymidine in the cytoplasm, thus limiting the availability of thymidine for phosphorylation by TK2 in mitochondria. The dTTP pool shows an exquisite sensitivity to variations of thymidine concentrations at the nanomolar level.     

Human thymidine kinase. Purification and properties of the cytosolic enzyme of placenta

Cytosolic thymidine kinase (EC 2.7.1.21) has been purified 5200-fold to apparent homogeneity from normal human placenta. The purification includes sequential affinity chromatography on blue-Sepharose and a thymidine column. The molecular weight of the enzyme determined by gel filtration and sucrose density ultracentrifugation is 92,000. The subunit molecular weight is 44,000, suggesting that the enzyme is a dimer in its native state. With isoelectric focusing, placental thymidine kinase demonstrated a single form with an isoelectric point of 9.1. The final purified enzyme preparation exhibits no immunological cross-reactivity with human mitochondrial thymidine kinase.     

Transition from cytosolic to mitochondrial thymidine kinase during development in human fetal tissues

The transition from cytosolic ("fetal") to mitochondrial ("adult") thymidine kinase, as detected by electrophoresis, was examined in six human fetal tissues of gestational ages 11-40 weeks. In all tissues there was an early period during development in which only the fetal form was detected, followed by a transitional period in which both fetal and adults forms were present, followed by a later period in which only the adult enzyme occurred. Transitional periods were 23-25 wk. gestational age for colon, 13-15 wk. for kidney, 18-20 wk. for liver, 14-18 wk. for lung, 34-36 wk. for serum, and 25-28 wk. for thyroid. In all cases, only the adult form was present by the time of birth and persisted during the first 18 months of extrauterine life. The adult form, but not the fetal form, was inhibited by dCTP.     

Effect of valine 106 on structure-function relation of cytosolic human thymidine kinase. Kinetic properties and oligomerization pattern of nine substitution mutants of V106.

Information on the regulation and structure-function relation of enzymes involved in DNA precursor synthesis is pivotal, as defects in several of these enzymes have been found to cause depletion or deletion of mitochondrial DNA resulting in severe diseases. Here, the effect of amino acid 106 on the enzymatic properties of the cell-cycle-regulated human cytosolic thymidine kinase 1 (TK1) is investigated. On the basis of the previously observed profound differences between recombinant TK1 with Val106 (V106WT) and Met106 (V106M) in catalytic activity and oligomerization pattern, we designed and characterized nine mutants of amino acid 106 differing in size, conformation and polarity. According to their oligomerization pattern and thymidine kinetics, the TK1 mutants can be divided into two groups. Group I (V106A, V106I and V106T) behaves like V106WT, in that pre-assay exposure to ATP induces reversible transition from a dimer with low catalytic activity to a tetramer with high catalytic activity. Group II (V106G, V106H, V106K, V106L and V106Q) behaves like V106M in that they are permanently high activity tetramers, irrespective of ATP exposure. We conclude that size and conformation of amino acid 106 are more important than polarity for the catalytic activity and oligomerization of TK1. The role of amino acid 106 and the sequence surrounding it for dimer-tetramer transition was confirmed by cloning the putative interface fragment of human TK1 and investigating its oligomerization pattern.     

Substrate specificity of three viral thymidine kinases (TK): vaccinia virus TK, feline herpesvirus TK, and canine herpesvirus TK

In search of novel suicide gene candidates we have cloned and characterized thymidine kinases from three viruses; vaccinia virus TK (VVTK), feline herpesvirus TK (FHV-TK), and canine herpesvirus TK (CHV-TK). Our studies showed that VVTK primarily is a thymidine kinase, with a substrate specificity mainly restricted to dThd and only minor affinity for dCyd. VVTK also is related closely to mammalian thymidine kinase 1 (TK1), with 66% identity and 75% general homology. Although CHV-TK and FHV-TK are sequence related to herpes simplex virus types 1 thymidine kinase (HSV1-TK), with 31% and 35% identity and a general similarity of 54%, the substrate specificity of these enzymes was restricted to dThd and thymidine analogs.     

Design, synthesis and enzymatic activity of highly selective human mitochondrial thymidine kinase inhibitors

Highly selective arabinofuranosyl nucleosides, which inhibit the mitochondrial thymidine kinase (TK-2) without affecting the closely related herpes simplex virus type 1 thymidine kinase (HSV-1 TK), varicella-zoster virus thymidine kinase (VZV-TK), cytosolic thymidine kinase (TK-1) or the multifunctional Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK), have been obtained. SAR studies indicate a close relation between the length of the substituent at the 2' position of the arabinofuranosyl moiety and the inhibitory activity.     

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 (approximately 35 mg) and truncated hTK1 (approximately 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 degrees C, respectively, when measured without adding substrates. The presence of substrates stabilized both hTK1W (DeltaT(m) ranging from 5.6 to 12.5 degrees C) and hTK1M (DeltaT(m) ranging from 0.8 to 5.3 degrees 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 microM and a k(cat) of 0.28 s(-1) for wild-type hTK1. The truncated hTK1M has a K(M) of 0.87 microM 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.