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)

Deoxythymidine kinases (EC 2.7.1.--) induced in HeLa TK- cells by Herpes simplex Type I and Type II viruses both had a requirement for divalent cations. The enzymes had the highest activities in the presence of Mg2+, followed by Mn2+, Ca2+, Fe2+, and in that order, whereas they were inactive in the presence of Zn2+ and Cu2+. The amount of Mg2+ required for optimal activity was dependent on the amount of ATP present, so that optimal activities were found when the concentration of Mg2+ was equal to that of ATP; an excess of Mg2+ inhibited the reaction. The activities of various nucleoside triphosphates as phosphate donors for Herpes simplex virus Type I deoxythymidine kinase were in the order: ATP = dATP = ara ATP greater than CTP greater than dCTP greater than UTP greater than dUTP greater than GTP greater than dGTP. Those for Herpes simplex virus Type II deoxythymidine kinase were in the order: CTP greater than dCTP = ara CTP greater than dATP greater than ATP greater than UTP greater than GTP greater than dUTP = dGTP. For both deoxythymidine kinases induced by Herpes simplex virus, the nucleoside triphosphates tested exerted cooperative effects. The Km values of ATP and CTP for the Herpes simplex virus Type I enzyme were 30 and 70 muM respectively; whereas those for the Herpes simplex virus Typr II enzyme were 140 and 450 muM. Studies on binding of various thymidine analogs with free 5'-OH to these deoxythymidine kinases indicated that 5-substituted ethyl-, vinyl-, allyl-, propyl-, iodo- and bromo-dUrd as well as iodo5 dCyd and bromo5 dCyd had good affinity to both enzymes. In contrast, vinyl5 Urd, iodo5 Urd and arabinosylthymidine had good affinity only to the Herpes simplex virus Type I enzyme but not to the Herpes simplex virus Type II deoxythymidine kinase. All of these thymidine analogs were competitive inhibitors, with KI values in the range of 0.25 to 1.5 muM. Herpes simplex virus Type I deoxythymidine kinase was less sensitive to either dTTP or iodo dUTP inhibition than Herpes simplex virus Type II. Both dThd and dCyd could serve as substrates and competed with each other for Herpes simplex viruses Type I and Type II induced kinases, but they differed in their Km values for these enzymes. The Km values of dThd and dCyd were 0.59 muM and 25 muM for Herpes simplex virus Type I deoxythymidine kinase; while they were 0.36 muM and 88 muM respectively for the Herpes simplex virus Type II enzyme.
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PMID:Deoxythymidine kinase induced in the HELA TK- cells by herpes simplex virus type I and type II. Substrate specificity and kinetic behavior. 18 65

Cytoplasmic and mitochondrial deoxythymidine kinase isozymes derived from the blast cells of acute myelocytic leukemia differ in their substrate specificity and kinetic behavior. These enzymes require divalent cations for their activity. The data suggest that the major role of idvalent cations is to chelate with ATP; the complex thus formed serves as the phosphate donor for the reaction. The activity of various triphosphate nucleosides as a phosphate donor for cytoplasmic deoxythymidine kinase is as follows: ATP = dATP greater than ara-ATP greater than GTP greater than CTP greater than dGTP = dCTP greater than dUTP, whereas for mitochondrial deoxythymidine kinase, the order of activity is ATP greater than CTP greater than UTP = dATP greater than ara-ATP greater than dGTP = dCTP greater than dUTP. Neither IdUTP nor dTTP could serve as a phosphate donor in the reaction catalyzed by either isozyme. From the many pyrimidine analogues tested for their binding affinity to each of these isozymes, I-dUrd and Br-dUrd had high good affinity which was equivalent to that of deoxythymidine. 5-Allyl-dUrd, 5-ethyl-dUrd, and 5-propyl-dUrd were only weakly bound to each isozyme. 5-I-dCyd, 5-Br-dCyd, dCyd, and 5-vinyl-dUrd were tightly bound to mitochondrial deoxythymidine kinase but not to the cytoplasmic isozyme. dTTP and I-dUTP are potent inhibitors of the reaction catalyzed by both isozymes. In contrast, dCTP and ara-CTP are potent inhibitors only of the mitochondrial isozyme, but not of the cytoplasmic isozyme. ATP-MG2+ acts as a sigmoidal substrate of the cytoplasmic isozyme with a"Km" of 0.22 mM, and as a regular substrate of the mitochondrial isozyme with a Km of 0.1 mM. Deoxythymidine acts as a regular substrate for both cytoplasmic and mitochondrial isozyme with a Km of 2.6 and 5.2 muM, respectively. Initial velocity as well as product inhibition studies suggest that the cytoplasmic isozyme catalyzes the reaction via a "sequential" mechanism. In contrast, mitochondrial deoxythymidine kinase catalyzes the reaction via a "ping-pong" mechanism.
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PMID:Human deoxythymidine kinase II: substrate specificity and kinetic behavior of the cytoplasmic and mitochondrial isozymes derived from blast cells of acute myelocytic leukemia. 106 65

Several hormones, including insulin, glucagon, and glucocorticoids, regulate the expression of the rate-limiting gluconeogenic enzyme, phosphoenolpyruvate carboxykinase [GTP: oxaloacetate carboxy-lyase (transphosphorylating); EC 4.1.1.32; PEPCK] in liver. In this report we demonstrate that retinoic acid (RA) also regulates PEPCK expression by inducing a 3-fold increase in the rate of transcription of the PEPCK gene. A RA response element located between -468 and -431 in the PEPCK promoter mediates a 7-fold increase in expression of a chimeric construct containing the basal PEPCK promoter ligated to the chloramphenicol acetyltransferase reporter gene. This element confers RA responsiveness through the heterologous thymidine kinase promoter and functions relatively independent of position and orientation. An 18-base-pair core sequence (-451 to -434) (i) mediates an effect of RA on PEPCK gene expression and contains motifs found in two other RA response elements; (ii) corresponds to AF1, an accessory factor element that is an integral component of the complex glucocorticoid response unit in the PEPCK gene promoter; (iii) is in a region involved in the developmental expression of the PEPCK gene; and (iv) shows homology to elements involved in the tissue-specific regulation of genes, including the hepatic apolipoprotein genes and the alpha 1-antitrypsin gene.
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PMID:A retinoic acid response element is part of a pleiotropic domain in the phosphoenolpyruvate carboxykinase gene. 184 96

An overview was presented of our approach of inhibition of de novo and salvage pathways in pyrimidine and purine metabolism. 1. Combination of acivicin, an inhibitor of de novo biosynthesis, and dipyridamole, a transport inhibitor, provided synergistic cytotoxicity in hepatoma and colon carcinoma cells. 2. AZT, a competitive inhibitor of the salvage enzyme, thymidine kinase, and 5-FU or MTX provided synergistic cytotoxicity in hepatoma 3924A. In human colon carcinoma HT-29 cells AZT and methotrexate yielded synergistic cytotoxicity and thymidine and hypoxanthine together provided protection from the action of these drugs. 3. These observations are significant because in rat hepatoma 3924A and in human cell lines HT-29, HL-60 and K562 thymidine kinase activity was 16- to 67-fold higher than that of dTMP synthase. Therefore, inhibition of dTMP synthase activity alone may provide poor responses because the salvage pathways can circumvent this block. 4. In leukemic patients treated with tiazofurin, an inhibitor of IMP dehydrogenase, the rate-limiting enzyme of GTP biosynthesis, and with allopurinol, which inhibits GPRT activity through raising plasma hypoxanthine levels, synergistic therapeutic results were obtained. The responses in sensitive patients entailed a decrease in IMP dehydrogenase activity and GTP concentration in leukemic cells and down-regulation of the ras and myc oncogenes. The down-regulation of the ras oncogene by tiazofurin through the decrease of GTP concentration has now been shown in K562, HL-60 and hepatoma cells and in patients with chronic granulocytic leukemia in blast crisis. Tiazofurin may be useful in studies on selective depression of the expression of the ras oncogene. 5. In 27 consecutive patients 50% responded positively to tiazofurin treatment. From this group, 10 out of 12 patients (83%) with chronic granulocytic leukemia in blast crisis responded to tiazofurin treatment.
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PMID:Regulation of de novo and salvage pathways in chemotherapy. 187 99

The carbocyclic analog of 2'-deoxyguanosine [(+-)-2-amino-1,9-dihydro-9-[(1 alpha,3 beta,4 alpha)-3-hydroxy-4-(hydroxymethyl)cyclopentyl]-6H-purine-6-one] (2'-CDG) is highly active in cell culture against strains S148 and E377 of herpes simplex virus type 1 (HSV-1), both of which code for thymidine kinase, and much less active against strain BW10168 which is deficient in this enzyme activity. Antiviral activity is associated primarily with the D-enantiomer; the L-enantiomer has much lower but significant activity. The metabolism of racemic 2'-CDG and its D- and L-enantiomers was studied in uninfected HEp-2 cells and in HEp-2 cells infected with the S148 or BW10168 strains of HSV-1. Nucleotides were separated by HPLC, and their elution was monitored by spectrophotometry. The chromatograms of extracts of cells infected with the S148 strain and treated with (+/-)-2'-CDG or D-2'-CDG included a new peak which appeared in the triphosphate region. This peak, the area of which exceeded that of the GTP peak, was shown to be due to the triphosphate of 2'-CDG. The new peak was not observed by HPLC of extracts of uninfected cells treated with (+/-)-2'-CDG or either of its enantiomers, cells infected with the S148 strain and treated with L-2'-CDG, or cells infected with the BW10168 strain and treated with (+/-)-2'-CDG or either of its enantiomers. The results were similar when these studies were performed with uninfected Vero cells and with Vero cells infected with strain S148 of HSV-1. In experiments with D-[8-3H]-2'-CDG, small amounts of phosphates of 2'-CDG could also be detected in uninfected HEp-2 cells and in cells infected with the BW10168 strain of HSV-1. Thus, 2'-CDG apparently is a good substrate for the virus-coded kinase and a very poor substrate for cellular phosphorylating enzymes. The selective phosphorylation of 2'-CDG by the virus-specific kinase presumably is critical for its antiviral activity as it is for that of acyclovir and other acyclic derivatives of guanine.
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PMID:Phosphorylation of the carbocyclic analog of 2'-deoxyguanosine in cells infected with herpes viruses. 217 31

The c-myc protooncogene has been implicated in control of growth and differentiation of mammalian cells. For instance, growth arrest is often preceded by reduction in c-myc mRNA and gene transcription. To elucidate the mechanisms of control of c-myc gene transcription, we have begun to characterize the interaction of nuclear factors with the 719-base-pair (bp) c-myc regulatory domain, located 1139-421 bp upstream of the P1 start site of the mouse gene. Nuclear extracts from exponentially growing WEHI 231 murine B-lymphoma cells formed multiple complexes in mobility-shift assays. Changes in complex distribution were observed in growth-arrested WEHI 231 cells, and a major site of this interaction mapped to a 21-bp sequence that is similar to the sequences recognized by the NF-kappa B family of proteins. Binding of NF-kappa B-like factors was demonstrated by oligonucleotide competition. Induction of complex formation upon 70Z/3 pre-B- to B-cell differentiation, enhancement of binding by GTP, and detergent-induced release of inhibitor protein suggested that NF-kappa B itself is one member of the family that can bind. Transfection of thymidine kinase-chloramphenicol acetyltransferase constructs containing the 21-bp c-myc sequence into Jurkat cells demonstrated increased chloramphenicol acetyltransferase activity upon phorbol ester and phytohemagglutinin treatment. These results suggest the involvement of NF-kappa B-like factors in the regulation of c-myc transcription.
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PMID:Interaction of an NF-kappa B-like factor with a site upstream of the c-myc promoter. 219

Although small in size (20 kDa), the vaccinia virus (VV) thymidine kinase protein (EC 2.7.1.21 TK) is a relatively complex enzyme which must contain domains involved in binding both substrates (ATP and thymidine) and a feedback inhibitor (dTTP), as well as sequences directing the association of individual protein monomers into a functional tetrameric enzyme. Alignment of predicted amino acid sequences of the thymidine kinase genes from a variety of sources was used to identify highly conserved regions as a first step toward locating potential regions housing essential domains. A conserved domain (domain I) near the amino terminus of VV TK protein had characteristics consistent with a nucleotide-binding site. Analysis of the nucleotide substrate specificity of VV TK indicated that ATP acts as the major phosphate donor for thymidine phosphorylation while GTP, CTP, and UTP were inefficient substrates. Site-directed mutagenesis was performed on domain I to generate 11 mutant enzymes. Comparison of the wild-type and mutant proteins with regard to enzyme activity revealed that two of the mutant enzymes, T18 and S19, exhibited enhanced enzyme activity (3.73-fold and 1.35-fold, respectively) relative to the control. The other mutations introduced led to greatly reduced levels of enzyme activity which correlated with a reduced or altered ability of the mutant enzymes to bind ATP as determined by ATP-agarose affinity chromatography. Wild-type VV TK bound to an ATP affinity column could also be eluted with dTTP. Glycerol gradient separation of wild-type TK in the presence or absence of dTTP indicated that dissociation of the tetrameric complex was not the means by which enzymatic inhibition was achieved. Taken together, these results suggest that (i) domain I (amino acids 11-22) of the VV TK corresponds to the ATP-binding site, and (ii) that dTTP is able to interfere with ATP binding, either directly or indirectly, and thereby inhibit enzymatic activity without dissociating the native enzyme.
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PMID:Identification of the ATP-binding domain of vaccinia virus thymidine kinase. 221 49

Acyclovir, an acrylic purine nucleoside analog, is a highly potent inhibitor of herpes simplex virus (HSV), types 1 and 2, and varicella zoster virus, and has extremely low toxicity for the normal host cells. This selectivity is due to the ability of these viruses to code for a viral thymidine kinase capable of phosphorylating acyclovir to a monophosphate; this capability is essentially absent in uninfected cells. The acyclovir monophosphate (acyclo-GMP) is subsequently converted to acyclovir triphosphate (acyclo-GTP) by cellular enzymes. Acyclo-GTP persists in HSV-infected cells for many hours after acyclovir is removed from the medium. The amounts of acyclo-GTP formed in HSV-infected cells are 40 to 100 times greater than in uninfected Vero cells. Acyclo-GTP acts as a more potent inhibitor of the viral DNA polymerases than of the cellular polymerases. The DNA polymerases of HSV-1 and HSV-2 also use acyclo-GTP as a substrate and incorporate acyclo-GMP into the DNA primer-template to a much greater extent than do the cellular enzymes. The viral DNA polymerase binds strongly to the acyclo-GMP-terminated template, and in thereby inactivated.
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PMID:Mechanism of action and selectivity of acyclovir. 628 36

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

Two uniquely paired deoxynucleoside kinases, deoxycytidine kinase/deoxyadenosine kinase (dCK/dAK) and deoxyguanosine kinase/deoxyadenosine kinase (dGK/dAK) are required, together with thymidine kinase (TK), for deoxynucleotide synthesis in Lactobacillus acidophilus R-26. Using polymerase chain reaction-generated probes based on N-terminal amino acid sequences, we have cloned tandem genes for 25- and 26-kDa polypeptides, whose derived amino acid sequences and size correspond to wild-type Lactobacillus enzyme subunits. Expression in Escherichia coli uses a single endogenous promoter and yields active dGK/dAK (approximately 3% of extracted protein) closely resembling wild-type dGK/dAK in specificity, kinetics, heterotropic activation, and end product inhibition. Alignment of cloned genes reveals 65% identity in their DNA sequences and 61% identity in derived amino acid sequences. Comparison with herpes-viral TKs reveals three conserved regions: glycine- and arginine-rich ATP-binding motifs and a D/E-R-S/H motif at the putative TK deoxynucleoside site. Greater homology, however, is seen upon multiple alignment of dGK with mammalian deoxycytidine kinases, yielding the consensus sequence-D/E-R-S-I/V-Y-x-D-.dGK also shares a sequence (-Y-D-P-T-I/L-E-D-S/Y-Y-) required for GTP hydrolysis by p21ras.
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PMID:Cloning and expression of the heterodimeric deoxyguanosine kinase/deoxyadenosine kinase of Lactobacillus acidophilus R-26. 789 98


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