Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

The distribution of the de novo and the salvage pathway enzymes for pyrimidine synthesis in the rat testis was investigated in separated seminiferous tubules and interstitial tissue. Aspartate carbamyltransferase (de novo synthesis) was localized primarily in the seminiferous tubules while the uridine and thymidine kinase activities were associated mainly with the interstitial tissue. To further delineate this distribution these enzymes were studied in testes from rats treated with Busulfan (the methane sulfonic acid diester of 1,4-butanediol), which causes degeneration of spermatogonia but not Sertoli cells. The results indicate that a part of the low tubular thymidine kinase activity resides in the germinal elements while the tubular uridine kinase activity is localized in the Sertoli cells.
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PMID:Distribution of pyrimidine synthetic enzymes in the rat testis. 51 6

Alignment of prokaryotic and vertebrate type II thymidine kinases (TK) (EC 2.7.1.21), such as that encoded by vaccinia virus (VVTK), reveals three conserved regions: designated domains I, III, and VII. Domains I and III of VVTK contain residues which closely resemble segments A (ATP) and B (Mg2+), respectively, of a Mg.ATP binding descriptor proposed previously (Walker, J.E., Saraster, M., Runswick, M-J., and Gay, N.J. (1982) EMBO J. 1, 945-951). In support of this hypothesis, domain I of the VVTK enzyme has previously been identified as the ATP binding site (Black and Hruby, 1990b). With regard to Mg2+ binding, several features of the VVTK domain III suggest that it may be responsible for this activity: 1) sequence similarity to a magnesium binding motif proposed previously (Walker, J.E., Saraster, M., Runswick, M-J., and Gay, N.J. (1982) EMBO J. 1, 945-951); 2) alignment of the predicted secondary structure of type II TK enzymes with other magnesium-binding enzymes such as adenylate kinase, EF-TU, and p21 reveals a conserved aspartic acid residue preceded by several hydrophobic residues with domain III; and 3) the conserved VVTK domain III aspartic acid residue (D82) aligns with D93 residue of adenylate kinase which is has been shown by NMR to participate in Mg2+ binding (Yan, H., and Tsai, M.-D., Biochemistry, in press). To directly examine the potential contribution of the conserved domain III D82 residue of VVTK in magnesium binding, site-directed mutagenesis was performed on positions D82 and G84 to generate four mutants, N82, L82, I82, and V84. Each mutant was analyzed for enzyme activity, divalent cation requirements, tetramer formation, and ATP binding ability. The results obtained were consistent with D82 playing a direct role in Mg2+ binding and suggest that while the aspartic acid does not appear to participate directly with ATP binding it may instead act to facilitate ATP hydrolysis by binding Mg2+ which aids to correctly position ATP for nucleophilic attack.
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PMID:Site-directed mutagenesis of a conserved domain in vaccinia virus thymidine kinase. Evidence for a potential role in magnesium binding. 155 87

Exploitation of differences in the substrate specificity of the type I and type II thymidine kinases (EC 2.7.1.21, TK) expressed by the Herpesviridae and Poxviridae (and human cells), respectively, has lead to the development of effective antiherpetic drugs such as acyclovir and gancyclovir. Analysis of type I TK protein sequences reveals a consensus sequence which corresponds to domain IV of type II TK proteins such as that encoded by vaccinia virus (VV). The type I descriptor (Xpho - + + Xpho) differs at the second position from the type II consensus sequence (Xpho Xphi + + Xpho) by having an aspartic acid residue (D) substituted for a glutamine (Q). To test the hypothesis that this substitution may be responsible for the observed differences in substrate specificity of these enzymes and as a approach to identify the nucleoside binding site of the type II VV TK, site-directed mutagenesis was employed to alter glutamine 114 (Q114) within domain IV of VV TK to a histidine (Q114H) or an aspartic acid (Q114D). All of the mutant enzymes retained full enzymatic activity as compared to wild-type VV TK when thymidine or bromodeoxyuridine were used as substrates. However, unlike the wild-type herpes simplex (type 1) TK enzyme, neither wild-type nor domain IV VV TK mutants were able to phosphorylate acyclovir or cytidine substrates. Surprisingly, the domain IV VVTK mutants displayed a dramatic loss of feedback inhibition by dTTP. Mutations of the Q114 position also lead to a difference in ATP binding as demonstrated by an altered elution pattern of Q114H and Q114D from an ATP-agarose affinity column with dTTP. Taken together, these results suggest that domain IV of VV TK is not involved directly in substrate discrimination but instead participates in feedback inhibition by dTTP.
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PMID:A single amino acid substitution abolishes feedback inhibition of vaccinia virus thymidine kinase. 157 11

The nucleotide sequences of the coding region of the thymidine kinase (TK) gene of pseudorabies virus strain NIA3 and a TK- mutant (ATK5) were determined. The coding region of the TK gene consists of 320 codons capable of producing a polypeptide with an Mr of 34979. The mutant expressed an inactive TK polypeptide when translated in vitro; a unique base substitution was detected in the mutant TK modifying amino acid position 13, at which aspartic acid replaces glycine. This modification affects the nucleotide-binding site, thus explaining the expression of an inactive TK polypeptide.
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PMID:Loss of pseudorabies virus thymidine kinase activity due to a single base mutation and amino acid substitution. 164 83

We have applied the polymerase chain reaction (PCR) technique to analyse mutations in the thymidine kinase (TK) gene of varicella-zoster virus (VZV) associated with resistance to the 5-bromovinyl (BVaraU) and 5-propynyl (PYaraU) analogues of arabinofuranosyl deoxyuridine. The results from this study allow three clear conclusions to be drawn. Firstly, the technique clearly shows that populations of VZV derived from plaque purification were truly clonal only when the plaques were initiated from cell-free virus (representing a tiny fraction of infectious virus) and plaques initiated by infected cells contained a mixture of variants. Secondly, despite the background mutations caused by errors of the Taq DNA polymerase, mutations relevant to drug resistance can easily be distinguished. The BVaraU-resistant mutant, 7-1, contained an aspartic acid to asparagine mutation at residue 18 and a single base deletion (position 65298 of the VZV DNA sequence), resulting in a frameshift and premature termination of the polypeptide chain, was found in the BVaraU-resistant mutant YSR. PYaraU-resistant virus populations contained viruses with one or more of three independent mutations, i.e. single base substitutions resulting in mutations from leucine to proline at residue 92, histidine to arginine at residue 97 and a deletion of 20bp (residues 65,135 to 65,154). Finally, the technique has uncovered novel sites in the virus TK associated with drug resistance. We conclude that in vitro amplification using the PCR combined with cloning and sequencing is a relatively rapid method for identifying mutations in small virus populations even when they are not homogeneous.
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PMID:Analysis of mutations in the thymidine kinase genes of drug-resistant varicella-zoster virus populations using the polymerase chain reaction. 184 97

Mycobacterium microti incorporated a wide range of exogenously supplied pyrimidines into its nucleic acids. M. avium incorporated a relatively narrow range of pyrimidines but both M. avium and M. microti when recovered after growth in vivo incorporated a slightly wider range of pyrimidines than the same strains grown in vitro. M. microti and M. leprae could not take up uridine nucleotides directly but could utilize the pyrimidines by hydrolysing them to uridine and then taking up the uridine. Pyrimidine biosynthesis, judged by the ability to incorporate carbon from CO2 or aspartate into pyrimidines was readily detected in non-growing suspensions of M. microti and M. avium harvested from Dubos medium, which does not contain pyrimidines. The biosynthetic activity was diminished in mycobacteria grown in vivo when there is likely to be a source of pyrimidines which they might use. Relative activities for pyrimidine biosynthesis de novo in M. microti were 100 for cells isolated from Dubos medium, 6 for cells isolated from Dubos medium containing the pyrimidine cytidine and 11 from cells recovered after growth in mice. In contrast, relative activities for a scavenging reaction, uracil incorporation, were 100, 71 and 59, respectively. Three key enzymes in the pathway of pyrimidine biosynthesis de novo were detected in M. microti and M. avium. Two, dihydroorotate synthase and orotate phosphoribosyltransferase appeared to be constitutive in M. microti and M. avium. Aspartate transcarbamoylase activity was higher in these mycobacteria grown in vivo than in Dubos medium but it was repressed in M. microti or M. avium grown in Dubos medium in the presence of 50 microM-pyrimidine. Aspartate transcarbamoylase was strongly inhibited by the feedback inhibitors ATP, CTP and UTP. Enzymes for scavenging pyrimidines were detected at low specific activities in all mycobacteria studied. Activities of phosphoribosyltransferases, enzymes that convert bases directly to nucleotides, were not related to the ability of intact mycobacteria to take up pyrimidine bases while activities of pyrimidine nucleoside kinases were generally related to the ability of intact mycobacteria to take up nucleosides. Phosphoribosyltransferase activity for uracil, cytosine, orotic acid and--in organisms grown in Dubos medium with 50 microM-uridine-thymine, as well as kinases for uridine, deoxyuridine, cytidine and thymidine were detected in M. microti. However, M. avium only contained uracil and orotate phosphoribosyltransferase, uridine, cytidine and thymidine kinase, and additionally deoxyuridine kinase when grown axenically with 50 microM-uracil, reflecting its more limited abilities in pyrimidine scavenging.
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PMID:Biosynthesis and scavenging of pyrimidines by pathogenic mycobacteria. 219 Oct 77

Previously constructed Swiss mouse 3T3 fibroblasts producing polyomavirus large T antigen after addition of dexamethasone were used to study the transcriptional activation by the viral protein of five genes coding for enzymes involved in DNA synthesis and precursor production, namely, dihydrofolate reductase, thymidine kinase, thymidylate synthase, DNA polymerase alpha, and proliferating-cell nuclear antigen. It was found that all these genes, whose expression is stimulated at the G1/S boundary of the cell cycle after growth stimulation by serum addition, are coordinately trans activated when T antigen is induced in cells previously growth arrested by serum withdrawal. Cell lines carrying the information for a mutant form of large T antigen, in which a glutamic acid residue in the binding site for the retinoblastoma protein was changed into aspartic acid, were constructed to test the involvement of an interaction of T antigen with the retinoblastoma protein in this reaction. It was found that the mutated T protein is incapable of stimulating transcription of any one of the genes. The promoter of three of the genes (dihydrofolate reductase, thymidine kinase, and DNA polymerase alpha) unequivocally carries binding sites for transcription factor E2F, suggesting that complexes forming with this growth- and cell cycle-regulating transcription factor are the targets for T antigen. Although there is so far no evidence that thymidylate synthase and proliferating cell nuclear antigen are regulated via E2F, our data indicate that the retinoblastoma protein still is involved in the control of these genes. mRNA for E2F itself increases in amount at the G1/S border in serum-stimulated cells but not during polyomavirus T antigen-induced transcriptional activation of DNA synthesis enzymes in arrested cells.
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PMID:Coordinated trans activation of DNA synthesis- and precursor-producing enzymes by polyomavirus large T antigen through interaction with the retinoblastoma protein. 790 59

The 545-residue Cln2 protein, like the other G1 cyclins of Saccharomyces cerevisiae, is a very unstable protein. This instability is thought to play a critical role in regulating cell cycle progression. The carboxyl-terminal domains of Cln2 and the other G1 cyclins contain sequences rich in Pro, Glu (and Asp), Ser, and Thr (so-called PEST motifs) that have been postulated to make up the signals that are responsible for the rapid degradation of these and other unstable proteins. To test this hypothesis, the carboxyl-terminal 178 residues of Cln2 were fused to the C terminus of a reporter enzyme, a truncated form of human thymidine kinase (hTK delta 40). The resulting chimeric protein (hTK delta 40-Cln2) retained thymidine kinase activity but was markedly less stable than hTK, hTK delta 40, or an hTK-beta-galactosidase fusion protein, as judged by enzyme assay, immunoblotting with anti-hTK antibodies, pulse-chase analysis of the radiolabeled polypeptides, and ability to support the growth of a thymidylate auxotroph (cdc21 mutant) on thymidine-containing medium. Thus, the presence of the Cln2 PEST domain was sufficient to destabilize a heterologous protein. Furthermore, the half-life of hTK delta 40-Cln2 was similar to that of authentic Cln2, and the rate of degradation of neither protein was detectably enhanced by treatments known to cause G1 arrest, including exposure of MATa haploids to alpha-factor mating pheromone and shifting cdc28ts and cdc34ts mutants to the restrictive temperature. These results suggest that the major signals responsible for Cln2 instability are confined to its C-terminal third. Because hTK delta 40-Cln2 and Cln2 were expressed from heterologous promoters yet their half-lives both in asynchronous cultures and when arrested at various cell cycle stages were always similar, the Cln2 PEST domain contains a signal for rapid protein turnover that is constitutively active and operative throughout the cell cycle. Removal of the 37 codons that encode the most prominent PEST-like segment from either hTK delta 40-Cln2 or Cln2 decreased the turnover rate of the resulting proteins, as expected; however, an hTK delta 40 chimera containing only this 37-residue segment was not detectably destabilized, suggesting that this PEST sequence, when removed from its normal context, is not a self-contained determinant of protein instability.
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PMID:G1 cyclin degradation: the PEST motif of yeast Cln2 is necessary, but not sufficient, for rapid protein turnover. 796 35

Despite the extensive use of antiviral drugs for the treatment of herpesvirus infections and as pro-drugs for ablative gene therapy of cancer, little structural information about the drug activating enzyme, herpes simplex virus type 1 thymidine kinase (TK), was available until recently. In the absence of the three-dimensional structure we sought to elucidate the function of the key aspartic acid residue (Dl62) present within a highly conserved tri-peptide motif that is thought to function in nucleoside binding. In this study we generated a mutant, D162Q, by site-directed mutagenesis, purified both the wild-type and mutant TKs to near homogeneity by single-step affinity chromatography and determined the kinetic parameters for thymidine, ATP, dTMP and dTTP interactions. A 12-fold increase in Km for thymidine by D162Q TK (Km = 6.67 microM) relative to wild-type enzyme (Km = 0.56 microM) was observed and the absence of any alteration in Km for ATP suggests that D162 participates in nucleoside binding. Furthermore, the Ki for dTMP is significantly higher for D162Q TK than for HSV-1 TK which is indicative of a shared or overlapping binding site with thymidine. This assessment is further supported by the different inhibition patterns of D162Q and wild-type TKs observed using [alpha-32P]5-N3dUMP photoaffinity labelling in the presence of thymidine, ganciclovir or dTMP. Interestingly, the Ki for dTTP was 30-fold lower for D162Q TK (Ki = 2.2 microM) than for the wild-type enzyme (Ki = 65.8 microM) which provides further evidence of the importance of D162 in TK function.
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PMID:Effect on substrate binding of an alteration at the conserved aspartic acid-162 in herpes simplex virus type 1 thymidine kinase. 875 95

Suicide gene therapy systems such as the herpes simplex thymidine kinase/ganciclovir system (TK/GCV) may kill cancer cells by apoptosis through as yet undefined mechanisms. Here we show that TK/GCV treatment induces p53 accumulation and increases cell surface expression of CD95 and tumor necrosis factor receptor, which is likely to involve p53-mediated translocation of CD95 to the cell surface. TK/GCV-induced apoptosis involves CD95-L-independent CD95 aggregation leading to the formation of a Fas-associated death domain protein (FADD) and caspase-8-containing, death-inducing signaling complex. Dominant negative FADD, the caspase-8 inhibitor zIETD-fmk [Z-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethylketone], and zVAD-fmk (Z-Val-Ala-Asp-fluoromethylketone) partially abrogate TK/GCV-induced apoptosis. In addition to apoptosis induction, TK/GCV treatment strongly sensitizes for CD95-L-, TNF-, and TNF-related, apoptosis-inducing, ligand (TRAIL)-induced cell death in constitutively resistant cells. These findings may be used to increase the efficacy of TK/GCV and other suicide gene therapy systems for the treatment of cancer.
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PMID:Herpes simplex virus thymidine kinase/ganciclovir-induced apoptosis involves ligand-independent death receptor aggregation and activation of caspases. 1041 38


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