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)

In Lactobacillus acidophilus R-26, the synthesis of DNA precursor deoxynucleotides occurs exclusively by salvage of deoxynucleosides, beginning with phosphorylation by four deoxynucleoside kinases. Subunits bearing three of these activities are uniquely organized into two heterodimers, deoxyadenosine/deoxycytidine kinase (dAK/dCK) and deoxyadenosine/deoxyguanosine kinase (dAK/dGK), which, along with a distinct deoxythymidine kinase (TK), catalyze the parallel first committed steps of dNTP biosynthesis. Whereas TK is common to most prokaryotes (and eukaryotes), the other three activities that are the emphasis of this review are quite unusual in bacteria. Each activity is regulated in cis by its homologous end-product (dNTP) which is understood to act as a multisubstrate inhibitor capable of binding to both nucleoside and phosphate subsites. Conversely, the inactive dAK subunit is progressively activated by 1) association with a dGK or dCK subunit and 2) the conformationally driven heterotropic affect of dGuo or dCyd bound to the opposing subunit. Limited proteolysis has proven to be a powerful probe of conformational states. Further indication of conformational or structural differences between dAK and dGK (or dCK) is that the former follows an ordered kinetic path, while dGK or dCK exhibits rapid-equilibrium random kinetics. The multi-substrate behavior of end-product binding provides a convenient new diagnostic tool for distinguishing kinetic mechanisms. Tandem dak-dgk genes have been cloned from Lactobacillus DNA and expressed in Escherichia coli as dAK/dGK, utilizing the associated promoter. Sequence alignments reveal 65% identity in their DNA and 61% in their derived amino acid sequences. Encoded N-terminal sequences are identical for the first 18 residues, and both subunits share conserved sequences in common with adenylate kinase and viral TK. A more unusual conserved element, which appears to play a role in the activation of dAK, resembles the G2 loop of p21 ras. Remarkably, no homologous gene(s) for the dAK/dCK pair could be found. Comparisons of amino acid sequences, isoelectric pHs and subunit masses strongly indicated that native dCK and dGK are identical in sequence, except at their extreme N-termini (M-IVL for dCK and -TVIVL for dGK), suggesting that processing of a common precursor occurs in Lactobacillus. Accordingly, deletion of codons 2 and 3 from dgk resulted in the expression of dAK/dCK in the E. coli host; its kinetic properties are indistinguishable from those of native dAK/dCK. Subcloning the dgk or engineered dck gene resulted in expression of active dGK or dCK homodimers, each with a virtually unchanged Km toward its primary deoxynucleoside. However, in common with human dCK, dCK (or dGK) homodimer exhibits secondary activities with much larger Kms towards dAdo and dGuo (or dCyd). dCTP (or dGTP) is the best inhibitor of all three activities of the respective homodimer. Fully active heterodimers can be reconstituted simply by mixing a homodimer with independently expressed (inactive) dAK.
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PMID:Life on the salvage path: the deoxynucleoside kinase of Lactobacillus acidophilus R-26. 942 44

Cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK) and the mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK) phosphorylate deoxynucleosides and their analogs. Recombinant human TK1 only phosphorylated beta-D Thd, but recombinant TK2, dCK and dGK all phosphorylated equally well beta-D and beta-L as well as to some extent alpha-D and alpha-L deoxynucleosides.
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PMID:The enantioselectivity of the cellular deoxynucleoside kinases. 1043 82

Deoxynucleoside kinases catalyze the 5'-phosphorylation of 2'-deoxyribonucleosides with nucleoside triphosphates as phosphate donors. One of the cellular kinases, deoxycytidine kinase (dCK), has been shown to phosphorylate several L-nucleosides that are efficient antiviral agents. In this study we investigated the potentials of stereoisomers of the natural deoxyribonucleoside to serve as substrates for the recombinant cellular deoxynucleoside kinases. The cytosolic thymidine kinase exhibited a strict selectivity and phosphorylated only beta-D-Thd, while the mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK) as well as dCK all had broad substrate specificities. TK2 phosphorylated Thd and dCyd stereoisomers in the order: beta-D- > or = beta-L- >> alpha-D- > or = alpha-L-isomer. dCK activated both enantiomers of beta-dCyd, beta-dGuo, and beta-dAdo with similar efficiencies, and alpha-D-dCyd also served as a substrate. dGK phosphorylated the beta-dGuo enantiomers with no preference for the ribose configuration; alpha-L-dGuo was also phosphorylated, and beta-L-dAdo and beta-L-dCyd were substrates but showed reduced efficiencies. The anomers of the 2',3'-dideoxy-D-nucleosides (ddNs) were tested, and TK2 and dCK retained their low selectivities. Unexpectedly, alpha-dideoxycytidine (ddC) was a 3-fold better substrate for dCK than beta-ddC. Similarly, alpha-dideoxythymidine (ddT) was a better substrate for TK2 than beta-ddT. dGK did not accept any D-ddNs. Thus, TK2, dCK, and dGK, similar to herpes simplex virus type 1 thymidine kinase (HSV-1 TK), showed relaxed stereoselectivities, and these results substantiate the functional similarities within this enzyme family. Docking simulations with the Thd isomers and the active site of HSV-1 TK showed that the viral enzyme may in some respects serve as a model for studying the substrate specificities of the cellular enzymes.
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PMID:Stereoisomeric selectivity of human deoxyribonucleoside kinases. 1060 35

The deoxynucleoside kinase reaction is often rate-limiting in the anabolism of pharmacologically active anti-cancer nucleosides. The levels of thymidine kinase (TK), deoxycytidine kinase, deoxyguanosine kinase (dGK), and thymidylate kinase were determined in leukocyte extracts from patients with chronic lymphocytic leukemia (CLL) and acute myelocytic leukemia (AML). The extracts from AML patients showed significantly higher TK activity than the ones from CLL patients. There were no differences in the levels of the other three kinases. In the case of dGK, the determinations were carried out with both an immunoblotting assay and selective enzyme activity measurements.
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PMID:Deoxynucleoside anabolic enzyme levels in acute myelocytic leukemia and chronic lymphocytic leukemia cells. 1127 69

Mitochondrial DNA (mtDNA)-depletion syndromes (MDS; OMIM 251880) are phenotypically heterogeneous, autosomal-recessive disorders characterized by tissue-specific reduction in mtDNA copy number. Affected individuals with the hepatocerebral form of MDS have early progressive liver failure and neurological abnormalities, hypoglycemia and increased lactate in body fluids. Affected tissues show both decreased activity of the mtDNA-encoded respiratory chain complexes (I, III, IV, V) and mtDNA depletion. We used homozygosity mapping in three kindreds of Druze origin to map the gene causing hepatocerebral MDS to a region of 6.1 cM on chromosome 2p13, between markers D2S291 and D2S2116. This interval encompasses the gene (DGUOK) encoding the mitochondrial deoxyguanosine kinase (dGK). We identified a single-nucleotide deletion (204delA) within the coding region of DGUOK that segregates with the disease in the three kindreds studied. Western-blot analysis did not detect dGK protein in the liver of affected individuals. The main supply of deoxyribonucleotides (dNTPs) for mtDNA synthesis comes from the salvage pathway initiated by dGK and thymidine kinase-2 (TK2). The association of mtDNA depletion with mutated DGUOK suggests that the salvage-pathway enzymes are involved in the maintenance of balanced mitochondrial dNTP pools.
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PMID:The deoxyguanosine kinase gene is mutated in individuals with depleted hepatocerebral mitochondrial DNA. 1168

The mitochondrial deoxyribonucleotide (dNTP) pool is separated from the cytosolic pool because the mitochondria inner membrane is impermeable to charged molecules. The mitochondrial pool is maintained by either import of cytosolic dNTPs through dedicated transporters or by salvaging deoxynucleosides within the mitochondria; apparently, enzymes of the de novo dNTP synthesis pathway are not present in the mitochondria. In non-replicating cells, where cytosolic dNTP synthesis is down-regulated, mtDNA synthesis depends solely on the mitochondrial salvage pathway enzymes, the deoxyribonucleosides kinases. Two of the four human deoxyribonucleoside kinases, deoxyguanosine kinase (dGK) and thymidine kinase-2 (TK2), are expressed in mitochondria. Human dGK efficiently phosphorylates deoxyguanosine and deoxyadenosine, whereas TK2 phosphorylates deoxythymidine, deoxycytidine and deoxyuridine. Here we identify two mutations in TK2, histidine 90 to asparagine and isoleucine 181 to asparagine, in four individuals who developed devastating myopathy and depletion of muscular mitochondrial DNA in infancy. In these individuals, the activity of TK2 in muscle mitochondria is reduced to 14-45% of the mean value in healthy control individuals. Mutations in TK2 represent a new etiology for mitochondrial DNA depletion, underscoring the importance of the mitochondrial dNTP pool in the pathogenesis of mitochondrial depletion.
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PMID:Mutant mitochondrial thymidine kinase in mitochondrial DNA depletion myopathy. 1168 1

Mycoplasmas are unable to synthesize purine and pyrimidine bases de novo. Therefore, salvage of existing nucleosides and bases is essential for their survival. Four mycoplasma species were studied with regard to their ability to phosphorylate deoxynucleosides. High levels of thymidine kinase (TK), deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK) and deoxyadenosine kinase (dAK) activities were detected in extracts from Mycoplasma pneumoniae, Mycoplasma mycoides subsp. mycoides SC (M. mymySC), Acholeplasma laidlawii (A. laidlawii) and Mycoplasma arginini (M. arginini). Nucleoside phosphotransferase activities were found at high levels in A. laidlawii and low levels in M. arginini. Pyrophosphate-dependent deoxynucleoside kinase activities were detected mainly in A. laidlawii and M. mymySC extracts. Two open reading frames were identified in the M. mymySC genome; one showed 25% sequence identity to human dGK and the other one had about 26% sequence identity to human TK1. The M. mymySC dGK-like enzyme was cloned, expressed in Escherichia coli and affinity-purified. This enzyme phosphorylated dAdo, dGuo and dCyd, and the highest catalytic rate was with dAdo as substrate. Therefore, we suggest that this enzyme should be named deoxyadenosine kinase. The physiological role of mycoplasma dAK and TK may be to support the unusually large dATP and dTTP pools required for replication of mycoplasma genomes.
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PMID:Novel deoxynucleoside-phosphorylating enzymes in mycoplasmas: evidence for efficient utilization of deoxynucleosides. 1173 47

In eukaryotic cells deoxyribonucleoside kinases belonging to three phylogenetic sub-families have been found: (i) thymidine kinase 1 (TK1)-like enzymes, which are strictly pyrimidine deoxyribonucleoside-specific kinases; (ii) TK2-like enzymes, which include pyrimidine deoxyribonucleoside kinases and a single multisubstrate kinase from Drosophila melanogaster (Dm-dNK); and (iii) deoxycytidine/deoxyguanosine kinase (dCK/dGK)-like enzymes, which are deoxycytidine and/or purine deoxyribonucleoside-specific kinases. We cloned and characterized two new deoxyribonucleoside kinases belonging to the TK2-like group from the insect Bombyx mori and the amphibian Xenopus laevis. The deoxyribonucleoside kinase from B. mori (Bm-dNK) turned out to be a multisubstrate kinase like Dm-dNK. But uniquely for a deoxyribonucleoside kinase, Bm-dNK displayed positive cooperativity with all four natural deoxyribonucleoside substrates. The deoxyribonucleoside kinase from X. laevis (Xen-PyK) resembled closely the human and mouse TK2 enzymes displaying their characteristic Michaelis-Menten kinetic with deoxycytidine and negative cooperativity with its second natural substrate thymidine. Bm-dNK, Dm-dNK and Xen-PyK were shown to be homodimers. Significant differences in the feedback inhibition by deoxyribonucleoside triphosphates between these three enzymes were found. The insect multisubstrate deoxyribonucleoside kinases Bm-dNK and Dm-dNK were only inhibited by thymidine triphosphate, while Xen-PyK was inhibited by thymidine and deoxycytidine triphosphate in a complex pattern depending on the deoxyribonucleoside substrate. The broad substrate specificity and different feedback regulation of the multisubstrate insect deoxyribonucleoside kinases may indicate that these enzymes have a different functional role than the other members of the TK2-like group.
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PMID:Deoxyribonucleoside kinases belonging to the thymidine kinase 2 (TK2)-like group vary significantly in substrate specificity, kinetics and feed-back regulation. 1181 27

Inorganic tripolyphosphate (PPP(i)) and pyrophosphate (PP(i)) were examined as potential phosphate donors for human deoxynucleoside kinase (dCK), deoxyguanosine kinase (dGK), cytosolic thymidine kinase (TK1), mitochondrial TK2, and the deoxynucleoside kinase (dNK) from Drosophila melanogaster. PPP(i) proved to be a good phosphate donor for dGK, as well as for dCK with dCyd, but not dAdo, as acceptor substrate, illustrating also the dependence of donor properties on acceptor. Products of phosphorylation were shown to be 5(')-phosphates. In striking contrast to ATP, the phosphorylation reaction follows strict Michaelis-Menten kinetics, with K(m) values of 74 and 92 microM for dCK and dGK, respectively, and V(max) values 40-50% that for ATP. With the other three enzymes, as well as for dCK with dAdo as acceptor, no, or only low levels (</=1% of that for ATP) of activity were observed. PP(i) was inactive (<0.1%) as a phosphate donor with all enzymes, but was a competitive inhibitor vs ATP, as was PPP(i) in systems with no or low donor activity. This is the first report on inorganic tripolyphosphate as a phosphate donor for nucleoside kinases, in particular human deoxyribonucleoside kinases.
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PMID:Inorganic tripolyphosphate (PPP(i)) as a phosphate donor for human deoxyribonucleoside kinases. 1253 61

In extension of an earlier report, six non-conventional analogues of ATP, three adenosine-2'-triphosphates (3'-deoxy, 3'-deoxy-3'-fluoro- and 3'-deoxy-3'-fluoroxylo-), and three adenosine-3'-triphosphates (2'-deoxy-, 2'-deoxy-2'-fluoro- and 2'-deoxy-2'-fluoroara-), were compared with ATP as potential phosphate donors for human deoxycytidine kinase (dCK), cytosolic thymidine kinase (TK1), mitochondrial TK2, deoxyguanosine kinase (dGK), and the deoxyribonucleoside kinase (dNK) from Drosophila melanogaster. With one group of enzymes, comprising TK1, TK2, dNK and dCK (with dAdo as acceptor), only 3'-deoxyadenosine-2'-triphosphate was an effective donor (5-60% that for ATP), and the other five analogues much less so, or inactive. With a second set, including dCK (dCyd, but not dAdo, as acceptor) and dGK (dGuo as acceptor), known to share high sequence similarity (approximately 45% sequence identity), all six analogues were good to excellent donors (13-119% that for ATP). With dCK and ATP1, products were shown to be 5'-phosphates. With dCK, donor properties of the analogues were dependent on the nature of the acceptor, as with natural 5'-triphosphate donors. With dCK (dCyd as acceptor), Km and Vmax for the two 2'(3')-deoxyadenosine-3'(2')-triphosphates are similar to those for ATP. With dGK, Km values are higher than for ATP, while Vmax values are comparable. Kinetic studies further demonstrated Michaelis-Menten (non-cooperative) or cooperative kinetics, dependent on the enzyme employed and the nature of the donor. The physiological significance, if any, of the foregoing remains to be elucidated. The overall results are, on the other hand, highly relevant to studies on the modes of interaction of nucleoside kinases with donors and acceptors; and, in particular, to interpretations of the recently reported crystal structures of dGK with bound ATP, of dNK with bound dCyd, and associated modeling studies.
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PMID:Striking ability of adenosine-2'(3')-deoxy-3'(2')-triphosphates and related analogues to replace ATP as phosphate donor for all four human, and the Drosophila melanogaster, deoxyribonucleoside kinases. 1274 3


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