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Query: EC:2.7.1.21 (thymidine kinase)
 7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Escherichia coli thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) is irreversibly inactivated by incubation with 3'-[3-(2-chloroethyl)-3-nitrosoureido]-3'-deoxythymidine (3'-CTNU). The inactivation of the enzyme followed first-order kinetics even after loss of 96% of the original activity. This indicates that the inactivation process is a one-kill phenomenon and not a generation of less active enzyme. The addition of a preincubated aqueous solution of 3'-CTNU to the enzyme reaction mixture did not inactivate the enzyme. ATP . Mg2+ but not thymidine protects the enzyme from inactivation by 3'-CTNU. The allosteric regulators, dTTP, dCTP and dCDP also afforded complete protection of the enzyme from inactivation by 3'-CTNU. These data indicate that the dimer form of the enzyme is completely resistant to inactivation by 3'-CTNU, but the monomer form of the enzyme is sensitive. The specificity of the protection is supported by the finding that neither ATP . Mg2+ nor thymidine protect yeast alcohol dehydrogenase from inactivation by this nitrosourea analog of thymidine.
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PMID:The effect of the nitrosourea analog of thymidine, 3'-[3-(2-chloroethyl)-3-nitrosoureido]-3'-deoxythymidine, on Escherichia coli thymidine kinase. 700 16

Syntheses are described of p1-(adenosine-5')-p3-(glucose-6) triphosphate (Ap3 glucose), Ap4 glucose, and p1-(adenosine-5')-P3-(thymidine-5') triphosphate (Ap3T). The compounds were not substrates of any of the enzymes used in the present studies. Ap3 glucose and Ap4 glucose were inhibitors of yeast hexokinase (HK) and the rat isozymes HK I-III; in general, they had less affinity for the enzymes than the substrates ATP and glucose. Inhibition constants (Ki values) of Ap3T with rat mitochondrial thymidine kinase (M-TK) and rat cytoplasmic TK (C-TK) were determined for variable thymidine (TdR) with a constant saturating level of ATP and for variable ATP with constant saturating TdR. Ap3T was a potent and selective inhibitor of M-TK [KM (TdR)/Ki = 1.6, KM (ATP)/Ki = 38 with variable ATP; KM (TdR) Ki = 0.06, KM (ATP)/Ki = 1.4 with variable TdR] relative to C-TK [KM (TdR)/Ki = 0.006, KM (ATP)/Ki = 0.7 with variable ATP; KM (TdR)/Ki = 0.001, KM (ATP)/Ki = 0.12 with variable TdR]. Inhibition of M-TK and C-TK by Ap3T differed qualitatively and quantitatively from inhibition under the same conditions by the metabolic feedback inhibitor TdR 5'-triphosphate.
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PMID:Species- or isozyme-specific enzyme inhibitors. 6. Synthesis and evaluation of two-substrate condensation products as inhibitors of hexokinases and thymidine kinases. 710 96

Cytosolic thymidine kinase (EC 2.7.1.75), the initial enzyme in the thymidine salvage pathway, was detected in crude homogenates of adult female Brugia pahangi and Dirofilaria immitis, with respective specific activities of 100 and 460 nmol/h/mg protein. Partially purified filarial thymidine kinases were found to have molecular weights of approximately 180 000, to be most active in the presence of Mg2+ and ATP, to have a sharp pH optimum (pH 7.0) and to be heat-labile in the absence of added thymidine. For both, the respective Km values for thymidine and ATP were 60 muM and 1.6 mM, and 5-iodo-2'-deoxyuridine was as good a substrate as thymidine. A distinguishing property was the 3-fold higher sensitivity of the B. pahangi enzyme to feedback inhibition by thymidine 5'-triphosphate. Adult female B. pahangi took up and incorporated [methyl-3 H] thymidine into DNA when they were exposed to this radiolabeled deoxynucleoside in vivo, but the thymidine salvage pathway in these worms was essentially nonfunctional in vitro.
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PMID:Thymidine kinase activity and thymidine salvage in adult Brugia pahangi and Dirofilaria immitis. 711 Jan 98

Thymidine kinase (ATP : thymidine 5'-phosphotransferase, EC 2.7.1.21), purified to apparent homogeneity from human liver, was found to have Michaelis constants for thymidine and ATP of 5 and 90 microM, respectively. Based on studies of initial velocity and product inhibition, the enzyme kinetic mechanism is compatible with an ordered sequential reaction with thymidine binding first and thymidine monophosphate released last. The activity of various triphosphate nucleosides as phosphate donors for human liver thymidine kinase showed little specificity with ATP greater than CTP greater than UTP greater than GTP and the respective Michaelis constants ranged from 0.10 to 0.30 mM. Among various purine and pyrimidine compounds, only TTp and dCTP were effective inhibitors of the enzyme. Inhibition with TTP was competitive with respect to both thymidine and ATP with Ki values of 13.5 and 8.5 microM, respectively, while the inhibition produced by dCTP was complex. Deoxycytidine was found to be an effective nucleoside substrate for human liver thymidine kinase with a Michaelis constant of 6 microM. This finding suggests that human mitochondrial deoxycytidine and thymidine kinase activity is a single protein.
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PMID:Kinetic mechanism and inhibition of human liver thymidine kinase. 728 1

Methyl benzimidazole carbamate and griseofulvin are two microtubule inhibitors which perturb plasmodial mitosis in Physarum polycephalum. These two compounds perturb not only the mitotic process leading to abortive mitosis but delay the onset of mitosis. Both drugs delayed the onset of thymidine kinase synthesis (EC 2.7.1.21, ATP: thymidine 5'phosphotransferase) which occurred concomitantly with mitosis. Thus, thymidine kinase synthesis, a cell cycle event which is not thought to have any relationship with the microtubular system, was perturbed in the same way as mitosis in the presence of two chemically unrelated microtubule inhibitors. It is suggested that the microtubular system is an essential part of a regulatory pathway of the cell cycle which plays a role in at least two events of the cell cycle: the onset of thymidine kinase synthesis, and the onset of mitosis.
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PMID:Regulation of thymidine kinase synthesis during the cell cycle of Physarum polycephalum: the effects of two microtubule inhibitors. 733 74

We have analyzed mutations in the thymidine kinase (TK) gene of varicella zoster virus (VZV) which showed resistance to 5-iodo-2'-deoxyuridine (IDU) and 5-bromo-2'-deoxyuridine (BrDU). Through sequencing of the TK gene, we found three amino acids were exchanged (41 Asn-->Ser, 266 Cys-->Ile, 288 Ser-->Leu). These mutations were not located at either the nucleoside- or the ATP-binding site. This result suggests that the resistance to IDU and BrDU in this particular strain is due to the change in conformation of TK rather than the replacement of amino acids in the binding sites.
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PMID:Analysis of mutations in the thymidine kinase gene of varicella zoster virus associated with resistance to 5-iodo-2'-deoxyuridine and 5-bromo-2'-deoxyuridine. 748 53

In mammalian cells, salvage pathway phosphorylation of thymidine is catalyzed by two thymidine kinases: the cell-cycle regulated cytoplasmic TK1 and the constitutively expressed mitochondrial TK2. Since TK1 is virtually absent in non-dividing cells, TK2 is probably the only thymidine kinase present in these cells. In cellular metabolism, TK1 and TK2 presumably serve to maintain sufficient dTTP for DNA replication and repair. TK1 purified from phytohemagglutinin-stimulated human lymphocytes is a dimer in the absence and a tetramer in the presence of ATP. In addition to the molecular weight transition, incubation with ATP at 4 degrees C or storage with ATP induces a reversible, enzyme concentration-dependent, kinetically slow transition from a low to a high affinity form of TK1, with Km values of 14 microM and 0.5 microM, respectively. This affinity difference implies that at cellular thymidine concentrations, the difference in catalytic activity between the two TK1 forms will be 3-5-fold. Calculations of cellular TK1 concentration suggested that the low affinity dimer form was dominant in G0/G1 cells and the high affinity tetramer form in S-phase cells. Hence, the transition may serve to fine-tune the cell-cycle regulation of thymidine kinase activity on the post-translational level. To study the ATP effect on the molecular level, an IPTG inducible T7 RNA polymerase-dependent expression system for the entire human TK1 polypeptide in E. coli was established. The recombinant TK1 has the same subunit mass and specific activity as the native enzyme. However, the recombinant TK1 solely displayed the kinetics of the high affinity form, with Km values of 0.3-0.4 microM regardless of pre-exposure to ATP, indicating that the ATP effect may be dependent on post-translational modifications absent in E. coli. Surprisingly, we did not observe any effect of ATP on TK1 purified from bone-marrow cells from a patient with acute monocytic leukemia (AMOL). Furthermore, the Km values of TK1 from these cells were 45 microM for the ATP-free enzyme and 65 microM for the ATP-incubated enzyme. With TK1 purified from HL-60 cells, we obtained the same pattern and kinetic values as for TK1 from lymphocytes. In the light of the results with the recombinant TK1, we presume that the lack of ATP effect and very high Km values observed for the AMOL TK1 may be due to changes in post-translational regulatory mechanisms in acute monocytic cells.
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PMID:Human thymidine kinase 1. Regulation in normal and malignant cells. 757 55

Previously we demonstrated that lines of transgenic mice carrying the herpes simplex type 1 virus thymidine kinase (HSV1-tk) reporter gene are male-sterile. Ectopic transcription of the HSV1-tk reporter in the testis was initiated downstream of the normal translation initiation codon and truncated proteins consistent with translational initiation at the second and third ATG codons were synthesized. Here we describe the effects on fertility 1) of converting the second and third ATG codons of the HSV1-tk reporter to CTG codons and 2) of utilizing the HSV type 2 thymidine kinase (HSV2-tk) reporter gene, in which the second ATG codon is located downstream of the ATP-binding pocket of the enzyme. Both reporters were coupled to the bovine thyroglobulin promoter (bTG-tk1 alpha and bTG-tk2 transgenes). The level of ectopic expression of these transgenes in the testis, relative to expression in the thyroid, was one to two orders of magnitude less than that of bTG-tk1. Sixty percent of male founders carrying the bTG-tk1 alpha and bTG-tk2 transgenes were fertile but did not transmit the transgene. In contrast, most males from subsequent generations were fertile and transmitted the transgenes at the expected frequency. This difference between founder males and male descendants is also observed with certain constructs in which the HSV1-tk reporter is coupled to other promoters. We attribute the effect to mosaicism among male founders, leading to competition between transgenic and nontransgenic spermatozoa and/or spermatogenic precursor cells and resulting in a lack of fertilization by transgenic sperm that would successfully fertilize eggs in the absence of competition.
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PMID:Different transmission rates of herpesvirus thymidine kinase reporter transgenes from founder male parents and male parents of subsequent generations. 757 10

Recombinant thymidine kinase from Herpes simplex virus type 1 (ATP:thymidine 5'-phosphotransferase; EC 2.7.1.21), an enzyme of therapeutic importance, was purified and crystallized in an N-terminally truncated but still fully active form. The three-dimensional structure was solved by X-ray diffraction analysis at 3.0 A resolution using isomorphous replacement. The chain fold is presented together with the bound substrates thymidine and ATP. Three chain segments at the surface could not be located. The chain fold, the location of the substrates and presumbly also the catalytic mechanism resemble the well-known adenylate kinases.
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PMID:The three-dimensional structure of thymidine kinase from herpes simplex virus type 1. 762 23

Recent results showed that ATP enables a kinetically slow shift from a low affinity form to a high affinity form of human cytosolic thymidine kinase (TK1), as reflected by the respective apparent Km values for thymidine of 15 microM and 0.7 microM. The shift is dependent on the concentration of enzyme protein, and calculations indicate that the low affinity form is predominant in G1 cells, and the high affinity form is predominant in S-phase cells. Here, we report that the two forms of TK1 differ manyfold in affinity to the substrate ATP, to the inhibitor dTTP and to various analogs of thymidine substituted in the pyrimidine or sugar. Furthermore, the kinetic reaction mechanisms suggest that the nucleoside analog. 3'-azidothymidine, used for treatment of infections with human immune deficiency virus (HIV), is not a substrate for the low affinity form of TK1.
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PMID:Different affinity of the two forms of human cytosolic thymidine kinase towards pyrimidine analogs. 763 20


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