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:3.5.4.4 (adenosine deaminase)
5,136 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have solved the structure of Escherichia coli cytidine deaminase (CDA) complexed to the transition state analog, 5-fluoroprimidin-2-one riboside. The monomer of the alpha 2 CDA dimer is composed of a small N-terminal alpha-helical domain with no obvious connection to the active sites, and two, larger, core domains. The two core domains have nearly identical tertiary structures and are related by approximate 2-fold symmetry, but lack internal amino acid sequence homology. Comparison of the core domain structure with known structures by sequence homology and structural compatibility searches suggests that the CDA tertiary structure cannot be superimposed on any known protein structure. The two active sites per dimer are formed across the subunit interface. The N-terminal core domain provides a pyrimidine nucleoside and zinc-binding pocket and the structurally homologous C-terminal core domain in the other monomer covers this active-site cleft, completely sequestering the ligand from solvent. The deeply buried zinc-binding site is formed by a novel "topological switch point" at the amino termini of two alpha-helices in consecutive alpha-beta-alpha-beta segments. The transition state analog is bound as a covalent hydrate at C4. The inhibitor hydroxyl oxygen atom interacts both with the zinc atom and the Glu104 carboxylate group, affording high differential affinity for the hydroxyl group relative to a hydrogen atom, in a manner reminiscent of that observed in adenosine deaminase (ADA). Unlike the latter enzyme, the zinc atom is coordinated in a tetrahedral ligand field to two cysteine and one histidine ligands, plus the hydroxyl group. Moreover, the inhibitor stereochemistry is of the opposite hand from that of the corresponding ADA inhibitor at C4(R), but is the same at the hydroxyl group O4(S). A consequence of these stereochemical differences is that in CDA a single conserved carboxylate side-chain, Glu104, can provide all of the necessary proton transfer functions involved in generating the zinc hydroxide nucleophile, and protonating the pyrimidine ring nitrogen atom and leaving amino group. The differences in zinc ligands, ligand-binding stereochemistry, and tertiary structures of CDA and ADA strongly suggest that the common features of transition state stabilization arose by convergent evolution.
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PMID:Cytidine deaminase. The 2.3 A crystal structure of an enzyme: transition-state analog complex. 828 86

The effect of hyperthermia on induction and repair of UV-radiation-induced cyclobutane pyrimidine dimers was investigated in the genome overall and in transcriptionally active and inactive genes in confluent human fibroblasts. Hyperthermia treatment (30 min, 45 degrees C) of human fibroblasts resulted in an increase in the protein content of isolated nuclei (protein aggregation) similar to that observed for HeLa S3 cells. The faster rate of disaggregation of nuclear proteins and the higher survival rate of heated fibroblasts in comparison with those for HeLa cells provide further evidence for a possible role of protein aggregation in heat-induced cell killing. Determination of the frequencies of cyclobutane pyrimidine dimers in the genome overall and in restriction fragments of the active adenosine deaminase (ADA) gene and inactive 754 locus revealed that hyperthermia selectively inhibits the induction of cyclobutane pyrimidine dimers in transcriptionally active DNA. Removal of cyclobutane pyrimidine dimers from the ADA gene was strongly delayed during the first 8 h in 10 J/m2 UV-irradiated fibroblasts. Such inhibition of repair of cyclobutane pyrimidine dimers was not observed for the 754 gene, indicating that inhibition of repair by hyperthermia is generally not mediated by inactivation of repair enzymes. It is proposed that the inhibition of induction and repair of cyclobutane pyrimidine dimers in active genes by hyperthermia is related to the heat-induced aggregation of proteins with the nuclear matrix, proximal to which active genes are located. Our results are consistent with a functional compartmentalization of DNA repair at the nuclear matrix.
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PMID:Heat-shock treatment selectively affects induction and repair of cyclobutane pyrimidine dimers in transcriptionally active genes in ultraviolet-irradiated human fibroblasts. 837 27

A new immunoextraction method using biotinylated antibodies and streptavidin-coated magnetic beads has been developed and applied to study the repair of uv-induced DNA damage in specific DNA sequences. uv-irradiated cells were allowed to carry out DNA repair for various time intervals in the presence of 5-bromodeoxyuridine (BrdU). Purified and restricted DNA was subjected to an immunoextraction method employing an anti-BrdU antibody (alpha Brdu), biotinylated goat antimouse antibodies (G alpha Mbio), and streptavidin-coated polymeric magnetic beads. Separation of BrdU containing DNA was achieved by using a magnetic device. This extraction procedure resulted in two fractions of DNA, i.e., BrdU-containing and non-BrdU-containing DNA. Both fractions were blotted on filters and subsequently hybridized with specific DNA probes to determine the relative amount of defined fragments in the two fractions of DNA. Repair experiments using normal primary human fibroblasts showed no difference in the incorporation of repair label in the active adenosine deaminase gene and the inactive 754 locus during the first 4 h following uv irradiation. After longer repair times the active gene incorporated more repair label than the inactive gene, consistent with the known preferential repair of cyclobutane pyrimidine dimers from active housekeeping genes.
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PMID:The use of streptavidin-coated magnetic beads and biotinylated antibodies to investigate induction and repair of DNA damage: analysis of repair patches in specific sequences of uv-irradiated human fibroblasts. 845 14

This study describes the induction and repair of UV-induced cyclobutane pyrimidine dimers (CPD) in transcriptionally active and inactive genes in the epidermis of the hairless mouse. Mice were exposed to a single dose of 2000 J/m2 ultraviolet B and kept in darkness for up to 24 h. The CPD frequency was measured in the transcriptionally active hypoxanthine-guanine phosphoribosyltransferase gene, the adenosine deaminase gene, the inactive c-mos protooncogene, and the haptoglobin gene using the CPD-specific enzyme T4 endonuclease V. Sixty % of the CPD was removed from the active genes during the first 4 h, after which no further repair took place up to 24 h. In contrast, the inactive genes did not show any removal of CPD. Assuming that the rate of repair in the c-mos and haptoglobin genes is representative for the repair rate in the genome overall, these results suggest only marginal repair of UV-induced CPD in the mouse epidermis in vivo. The selective repair of active genes in the epidermis of mice resembles that of rodent cells in culture and shows the biological relevance of repair studies performed with cultured rodent cells in vitro.
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PMID:Ultraviolet-induced cyclobutane pyrimidine dimers are selectively removed from transcriptionally active genes in the epidermis of the hairless mouse. 845 36

1. A newly found action of adenosine in neurons, which may have an important physiological function in the growth and development of the sympathetic nervous system, is described. Adenosine (1-100 microM) inhibited neurite outgrowth within the first 24 h and killed about 80% of sympathetic neurons supported by nerve growth factor over the next 2 days in culture. Neurons supported by excess KCl, forskolin or phorbol 12,13-dibutyrate were equally susceptible to the toxic actions of adenosine. Inosine, guanosine or hypoxanthine (all 100-300 microM) were without effect on neuronal growth and survival. 2. Specific agonists of adenosine A1 and A2 receptors were not neurotoxic, and toxic effects of adenosine were not antagonized by aminophylline. These results rule out involvement of adenosine receptors and the adenylyl cyclase-cAMP signalling system in neurotoxic actions of adenosine. 3. Adenosine toxicity was prevented by inhibitors of the adenosine membrane transporter, suggesting an intracellular site of action of adenosine. 4. Inhibitors of adenosine deaminase dramatically facilitated the toxic action so that physiologically relevant concentrations of adenosine were neurotoxic. 5. Adenosine kinase activity of sympathetic neurons was dose-dependently inhibited by 5'-iodotubercidin (3-100 nM). 5'-Iodotubercidin (100 nM) completely protected neurons against toxicity of adenosine plus adenosine deaminase inhibitors. These results provide convincing evidence that phosphorylation of the nucleoside is an essential requirement for initiation of adenosine toxicity. 6. Sympathetic neurons were successfully rescued from the lethal effects of adenosine deaminase inhibitor plus adenosine by uridine or 2-deoxycytidine, but not by nicotinamide or 2-deoxyguanosine, suggesting that depletion of pyrimidine nucleotides by phosphorylated adenosine compounds and consequent inhibition of DNA synthesis produces neuronal death. 7. DNA fragmentation, assessed by the fluorescent dye bisbenzimide and by the TUNEL (terminal deoxynucleotidyl transferase-mediated nick end labelling) method, indicated that neuronal death induced by adenosine was apoptotic. 8. We conclude that adenosine deaminase and adenosine kinase play an important role in the metabolism of intracellular concentrations of adenosine and thereby regulate the growth and development of sympathetic neurons. Our study highlights, for the first time, the importance of adenosine as a mediator of programmed cell death of neurons supported by nerve growth factor.
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PMID:Adenosine-induced apoptosis in chick embryonic sympathetic neurons: a new physiological role for adenosine. 856 48

Two of the hallmarks of Cockayne's syndrome (CS) are the hypersensitivity of cells to UV light and the lack of recovery of the ability to synthesize RNA following exposure of cells to UV light, in spite of the normal repair capacity at the overall genome level. The prolonged repressed RNA synthesis has been attributed to a defect in transcription-coupled repair, resulting in slow removal of DNA lesions from the transcribed strand of active genes. This model predicts that the sensitivity of CS cells to another DNA-damaging agent, i.e., the UV-mimetic agent N-acetoxy-2-acetylaminofluorene (NA-AAF), should also be associated with a lack of resumption of RNA synthesis and defective transcription-coupled repair of NA-AAF-induced DNA adducts. We tested this by measuring the rate of excision of DNA adducts in the adenosine deaminase gene of primary normal human fibroblasts and two CS (complementation group A and B) fibroblast strains. High-performance liquid chromatography analysis of DNA adducts revealed that N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) was the main adduct induced by NA-AAF in both normal and CS cells. No differences were found between normal and CS cells with respect to induction of this lesion either at the level of the genome overall or at the gene level. Moreover, repair of dG-C8-AF in the active adenosine deaminase gene occurred at similar rates and without strand specificity in normal and CS cells, indicating that transcription-coupled repair does not contribute significantly to repair of dG-C8-AF in active genes. Yet CS cells are threefold more sensitive to NA-AAF than are normal cells and are unable to recover the ability to synthesize RNA. Our data rule out defective transcription-coupled repair as the cause of the increased sensitivity of CS cells to DNA-damaging agents and suggest that the cellular sensitivity and the prolonged repressed RNA synthesis are primarily due to a transcription defect. We hypothesize that upon treatment of cells with either UV or NA-AAF, the basal transcription factor TFIIH becomes involved in nucleotide excision repair and that the CS gene products are involved in the conversion of TFIIH back to the transcription function. In this view, the CS proteins act as repair-transcription uncoupling factors. If the uncoupling process is defective, RNA synthesis will stay repressed, causing cellular sensitivity. Since transcription is essential for transcription-coupled repair, the CS defect will affect those lesions whose repair is predominantly transcription coupled, i.e., UV-induced cyclobutane pyrimidine dimers.
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PMID:The sensitivity of Cockayne's syndrome cells to DNA-damaging agents is not due to defective transcription-coupled repair of active genes. 875 44

Abnormalities in erythrocyte nucleotide metabolism are associated with hereditary nonspherocytic hemolytic anemia. Deficiency of adenylate kinase and pyrimidine 5'-nucleotidase and hyperactivity of adenosine deaminase shorten the red cell lifespan. Deficiency of adenylate kinase has been reported in four different families. Although in one family, total absence of this enzymatic activity was documented in one hematologically normal sibling, there is doubt about the capacity of this single enzyme deficiency to produce hemolysis. A deficiency of pyrimidine 5'-nucleotidase is a cause of hemolytic anemia characterized by red cells with basophilic stippling. This enzyme has been reported to catalyze the hydrolytic dephosphorylation of pyrimidine 5'-ribose monophosphate. Red cells of patients contain an increased concentration of pyrimidine nucleotides and reduced form of glutathione. In hyperactivity, the adenosine deaminase activity in erythrocytes may be increased to 100 times the normal level. The high adenosine deaminase activity of erythrocytes depletes adenine nucleotides, inhibiting its metabolism.
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PMID:[Hemolytic anemia due to abnormalities in erythrocyte nucleotide metabolism]. 889 May 81

We show here that 2'-deoxyadenosine (2'-dAdo) but not adenosine was toxic to chromaffin cells of 3-4-week-old rat adrenal glands. More than 75% of the cells plated in culture gradually died over a 3-day period in the presence of 100 microM 2'-dAdo plus 3 microM deoxycoformycin (DCF). Morphological observations together with bisbenzimide staining and terminal deoxynucleotidyl transferase-mediated nick and labeling showed membrane blebbing, shrinkage of cell bodies, chromatin condensation, and DNA fragmentation, suggesting apoptosis-like cell death by 2'-dAdo. Lethal effects of 2'-dAdo were potentiated by DCF, a drug that inhibits adenosine deaminase. 2'-dAdo-prompted cell death was not prevented by inhibitors of nucleoside transporter (3 microM dilazep or 1 microM nitrobenzylthioinosine), precursors of pyrimidine nucleotide biosynthesis (300 microM uridine or 100 microM 2'-deoxycytidine), or 5 mM nicotinamide. Cells incubated with 2'-dAdo (100 and 300 microM) showed a three- and ninefold, respectively, increase in content of dATP, a product known to be an inhibitor of ribonucleotide reductase, an enzyme essential for DNA synthesis. Formation of dATP was completely prevented by iodotubercidin (ITu), a drug that inhibits phosphorylation of 2'-dAdo to dATP by nucleoside kinase. It is interesting that nanomolar concentrations of ITu also completely protected chromaffin cells from 2'-dAdo lethality. Our study demonstrates for the first time that mammalian adrenal chromaffin cells undergo apoptotic cell death by a natural nucleoside and suggests that this model could be used to study apoptosis and cell function.
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PMID:2'-deoxyadenosine induces apoptosis in rat chromaffin cells. 893 58

Crystal structures of the cytidine deaminase-uridine product complex prepared either by cocrystallizing enzyme with uridine or by diffusing cytidine into ligand-free crystals show that the product binds as a 4-ketopyrimidine. They reveal four additional features of the catalytic process. (1) A water molecule bound to a site previously observed to bind the incoming 4-NH2 group represents the site for the leaving ammonia molecule. The conserved Pro 128 accommodates both moieties by orienting the carbonyl group of the previous residue. (2) The Glu 104 carboxylate group rotates from its hydrogen bond to the O4 hydroxyl group in transition-state analog complexes, forming a new hydrogen bond to the leaving group moiety. Thus, after stabilizing the hydroxyl group in the transition state, Glu 104 transfers a proton from that group to the leaving amino group, promoting enol-to-keto isomerization of the product. (3) Difference Fourier comparisons with transition-state complexes indicate that the pyrimidine ring rotates toward the zinc by approximately 10 degrees. The active site thus "pulls" the ring and 4-NH2 group in opposite directions during catalysis. To preserve coplanarity of the 4-keto group with the pyrimidine ring, the N1-C1' glycosidic bond bends by approximately 19 degrees out of the ring plane. This distortion may "spring-load" the product complex and promote dissociation. Failure to recognize a similar distortion could explain an earlier crystallographic interpretation of the adenosine deaminase-inosine complex [Wilson, D. K., & Quiocho, F. A. (1994) Nat. Struct. Biol. 1, 691-694]. (4) The Zn-Sgamma132 bond, which lengthens in transition-state complexes, shortens as the O4 atom returns to a state of lower negative charge in the planar product, consistent with our previous proposal that this bond buffers the zinc bond valence, compensating buildup of negative charge on the oxygen nucleophile during catalysis.
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PMID:The structure of the cytidine deaminase-product complex provides evidence for efficient proton transfer and ground-state destabilization. 912 97

Recently, we have demonstrated that adenosine and 2'-deoxyadenosine are toxic to embryonic sympathetic neurons and proposed that purine and pyrimidine metabolism may play a critical role in the growth and development of sympathetic neurons. To extend this hypothesis further, we examined the effects of these nucleosides on two other neuronal populations in the chick embryo, sensory dorsal root ganglion neurons and parasympathetic ciliary ganglion neurons. Now, we show that 2'-deoxyadenosine and adenosine have no visible adverse effect on the viability of either sensory or parasympathetic neurons. Instead, 2'-deoxyadenosine proved to be highly toxic to the nonneuronal cells. The toxic effects of 2'-deoxyadenosine were markedly enhanced by inhibition of adenosine deaminase. In contrast, adenosine was much less toxic to nonneuronal cells than 2'-deoxyadenosine and its effect was not potentiated by inhibition of adenosine deaminase. Priming of pyrimidine pools by exogenous uridine and the specific inhibitor of the nucleoside transporter, nitrobenzylthioinosine, did not protect nonneuronal cells from 2'-deoxyadenosine toxicity. Since phosphorylation of internalized nucleosides was a key step in the initiation of toxicity in sympathetic neurons, adenosine kinase activity was compared in sensory and sympathetic neuronal cultures. The adenosine kinase activity in dorsal root ganglion cultures was only 20% of that in sympathetic ganglion cultures. Furthermore, inhibition of phosphorylation by blocking 2'-deoxyadenosine kinase with iodotubercidin and 5'-amino-5'-deoxyadenosine had no protective effect against 2'-deoxyadenosine toxicity. [3H]-thymidine incorporation was inhibited over 90% by 2'-deoxyadenosine as early as 6 h following its addition and for up to 4 days, suggesting inhibition of proliferation of nonneuronal cells by 2'-deoxyadenosine. The nucleoside was also able to wipe out already well established nonneuronal cells, leaving behind an enriched population of sensory neurons. The selective vulnerability of nonneuronal cells to 2'-deoxyadenosine offers a convenient and effective tool for removing nonneuronal cells from neuronal cultures as well as providing a new model for studying the mechanisms of nucleoside toxicity.
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PMID:2'-Deoxyadenosine selectively kills nonneuronal cells without affecting survival and growth of chick dorsal root ganglion neurons. 955 58


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