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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Guanylyltransferase that catalyzes mRNA capping by the reaction, ppNpN + GTP----GpppNpN was purified from S. cerevisiae. The enzyme forms a nucleotidyl intermediate by phosphoamide linkage of GMP. Two guanylylated polypeptides of MR approximately 52,000 and 46,000 were obtained, the latter apparently by proteolysis of the larger component. Both forms transferred the covalently bound GMP to ppApG, yielding GpppApG. Dinucleoside tri- and tetraphosphates of the type Gp3N and Gp4N were also produced by using ribonucleoside 5'-di and triphosphates as acceptors. The purified yeast guanylyltransferase contained little or no RNA 5'-triphosphatase or methyltransferase.
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PMID:Synthesis of Gp4N and Gp3N compounds by guanylyltransferase purified from yeast. 632 12

GTP:mRNA guanylyltransferase, an enzyme that catalyzes the transfer of a GMP residue from GTP to the 5' end of RNA to form a cap structure identified as G(5')pppN-, has been isolated from HeLa cell nuclei. The enzyme has been purified approximately 1000-fold and separated by column chromatography (using DEAE-cellulose, phosphocellulose, Cibacron blue-agarose, and GTP-agarose) from a variety of other activities, including RNA triphosphatase and mRNA (guanine-7)methyltransferase. The reaction product was identified by its resistance to Penicillium nuclease and alkaline phosphatase, sensitivity to venom phosphodiesterase, and electrophoretic and chromatographic mobilities relative to authentic standards. Optimal enzyme activity was obtained at pH 7.5 in the presence of Mn2+ or Mg2+, GTP, and an appropriate acceptor polyribonucleotide. The enzyme was inhibited by elevated concentrations of salt and by sulfhydryl-binding reagents but was unaffected by S-adenosylmethionine or S-adenosylhomocysteine. A molecular weight of 48,500 was estimated by sucrose gradient centrifugation of purified enzyme.
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PMID:Purification and characterization of mRNA guanylyltransferase from HeLa cell nuclei. 735 12

Vaccinia virus mRNA capping enzyme is a multifunctional protein with RNA triphosphatase, RNA guanylyltransferase, RNA (guanine-7) methyltransferase, and transcription termination factor activities. The protein is a heterodimer of 95- and 33-kDa subunits encoded by the vaccinia virus D1 and D12 genes, respectively. The capping reaction entails transfer of GMP from GTP to the 5'-diphosphate end of mRNA via a covalent enzyme-(lysyl-GMP) intermediate. The active site is situated at Lys-260 of the D1 subunit within a sequence element, KxDG (motif I), that is conserved in the capping enzymes from yeasts and other DNA viruses and at the active sites of covalent adenylylation of RNA and DNA ligases. Four additional sequence motifs (II to V) are conserved in the same order and with similar spacing among the capping enzymes and several ATP-dependent ligases. The relevance of these common sequence elements to the RNA capping reaction was addressed by mutational analysis of the vaccinia virus D1 protein. Nine alanine substitution mutations were targeted to motifs II to V. Histidine-tagged versions of the mutated D1 polypeptide were coexpressed in bacteria with the D12 subunit, and the His-tagged heterodimers were purified by Ni affinity and phosphocellulose chromatography steps. Whereas each of the mutated enzymes retained triphosphatase, methyltransferase, and termination factor activities, six of nine mutant enzymes were defective in some aspect of transguanylylation. Individual mutations in motifs III, IV, and V had distinctive effects on the affinity of enzyme for GTP, the rate of covalent catalysis (EpG formation), or the transfer of GMP from enzyme to RNA. These results are concordant with mutational studies of yeast RNA capping enzyme and suggest a conserved structural basis for covalent nucleotidyl transfer.
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PMID:Mutational analysis of mRNA capping enzyme identifies amino acids involved in GTP binding, enzyme-guanylate formation, and GMP transfer to RNA. 756 75

Transcription termination by vaccinia virus RNA polymerase during synthesis of early mRNAs requires a virus-encoded termination factor (VTF). VTF is but one of many activities associated with the vaccinia virus mRNA capping enzyme, a heterodimer of 95- and 33-kDa subunits encoded by the D1 and D12 genes, respectively. Although the three catalytic domains involved in cap formation have been assigned to individual subunits or portions thereof, the structural requirements for VTF activity are unknown. We now report that both full-length subunits are required for transcription termination. The 844-amino acid D1 subunit by itself, which is fully active in triphosphatase and guanylyltransferase functions, has no demonstrable VTF activity in vitro. Neither does the D12 subunit by itself. The heterodimeric methyltransferase domain of D1 (residues 498 to 844) and D12 subunits also has no VTF activity. VTF is not affected by a K-to-M mutation of the guanylyltransferase active site at position 260 (K260M) that abolishes enzyme-GMP complex formation or by a H682A/Y683A double mutation of the D1 subunit, which abrogates methyltransferase activity. Thus, the structural requirements for termination are distinct from those for nucleotidyl transfer and methyl transfer.
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PMID:The D1 and D12 subunits are both essential for the transcription termination factor activity of vaccinia virus capping enzyme. 774 34

The genome of flaviviruses consists of an infectious single-stranded RNA molecule which contains a type 1 cap structure at the 5'-terminus. The cap is synthesized by RNA triphosphatase, guanylyltransferase and methyltransferase. Since flaviviruses replicate in the cytoplasm, it can be assumed that these functions are performed by virus-coded proteins. We previously showed that subtilisin treatment of membranes isolated from cells infected with the West Nile flavivirus results in release of a 50 kDa molecular weight fragment of the viral nonstructural protein NS 3. This so-called p50-S protein contains the residue gly (168) of NS 3 at the amino-terminus and represents an RNA-stimulated NTPase. In the present report we present experimental evidence which indicates that the p50-S protein also contains the active site of an RNA triphosphatase. The activity specifically cleaves the beta,gamma-triphosphate bond at the 5'-terminus of RNA. The localization of NS 3 protein sequence elements with known functions indicates that this multifunctional protein contains a protease in the amino-terminal part, a helicase in the central region and the RNA triphosphatase in the carboxy-terminal domain. An amino acid sequence element which may be involved in recognition of the 5'-terminal RNA triphosphate is tentatively identified. A homologous element may be present in the vaccinia virus-coded RNA triphosphatase.
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PMID:The NS 3 nonstructural protein of flaviviruses contains an RNA triphosphatase activity. 821 62

The vaccinia virus mRNA capping enzyme is a heterodimeric protein containing subunits of 97 and 33 kDa, the products of genes D1R and D12L, respectively. The enzyme catalyzes the first three reactions in the mRNA cap formation pathway: mRNA triphosphatase, guanyltransferase and (guanine-7-)methyltransferase. The guanyltransferase reaction proceeds by way of a covalent enzyme GMP (E-GMP) intermediate (Shuman, S. and Hurwitz, J. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 187-191) in which the GMP is linked to the large subunit through a lysine residue (Toyama, R., Mizumoto, K., Nakahara, Y., Tatsuno, T., and Kaziro, Y. (1983) Eur. J. Biochem. 2, 2195-2201; Roth, M. J., and Hurwitz, J. (1984) J. Biol Chem. 259, 13488-13494). In order to identify the map position of the guanyltransferase active site lysine residue, high specific activity [32P]E-GMP was prepared. Digestion of the E-GMP with hydroxylamine at pH 9.5 yielded a 31-kDa radioactive fragment derived from amino acids 1-273. Cleavage of E-GMP with cyanogen bromide produced a radioactive peptide of 14 kDa corresponding to amino acids 242-365. Lysine residues are found at positions 244 and 260. Staphylococcus aureus V8 protease digestion of cyanogen bromide-cleaved E-GMP yields a radioactive product of about 5 kDa in molecular mass corresponding to the peptide generated by cleavage at glutamic acid residues 253 and 297, demonstrating that lysine 260 is the site of linkage of GMP.
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PMID:Identification of the vaccinia virus mRNA guanyltransferase active site lysine. 822 60

Vaccinia virus capping enzyme, a heterodimer of 95-kDa and 33-kDa subunits, modifies the 5' RNA end and also acts as a transcription termination factor during synthesis of viral early mRNAs. Termination occurs in response to a specific signal, UUUUUNU, in the nascent RNA chain. We now report that purified capping enzyme binds to defined RNAs in solution to form complexes that are stable during native gel electrophoresis. Multiple enzyme molecules can bind to a single RNA. No particular 5' end structure is required for RNA binding, suggesting that the observed protein-RNA interaction is unrelated to the triphosphatase, guanylyltransferase, or methyltransferase functions of capping enzyme. Although binding does not require a UUUUUNU element in the RNA, complex formation is competed preferentially by poly(U) compared to poly(C). Capping enzyme binds to the synthetic 30-mer homopolymers to form a single protein-RNA complex; affinity for U-30 is 10-fold higher than for A-30. The sites of protein-RNA contact, as detected by UV cross-linking, are located predominantly within the 95-kDa capping enzyme subunit, which is itself sufficient to bind and cross-link to RNA in the absence of the small subunit.
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PMID:RNA binding properties of vaccinia virus capping enzyme. 840 63

The RNA 5'-triphosphatase, nucleoside triphosphate phosphohydrolase, and guanylyltransferase activities of the vaccinia virus mRNA capping enzyme were previously localized to an NH2-terminal 60-kDa domain of the D1R subunit. Measurement of the relative ATPase and guanylyltransferase activities remaining in D1R carboxyl-terminal deletion variants expressed in Escherichia coli BL21(DE3)plysS localizes the carboxyl terminus of the active domain to between amino acids 520 and 545. Failure to obtain a deletion mutant with the loss of one activity indicates that the catalysis of both reactions requires a common domain structure. Based on these results, a truncated D1R protein terminating at amino acid 545 was expressed in E. coli and purified to homogeneity. D1R1-545 was found to be kinetically equivalent to the holoenzyme in regard to ATPase, RNA 5'-triphosphatase, and guanylyltransferase activities. Measurement of RNA binding by mobility shift and UV photo-cross-linking analyses also demonstrates the ability of this domain to bind RNA independent of the methyltransferase domain, comprised of the carboxyl terminus of D1R from amino acids 498-844 and the entire D12L subunit. RNA binding to D1R1-545 is substantially weaker than binding to either the methyltransferase domain or the holoenzyme. Binding is inhibited by 5'-OH RNA and to a lesser extent by DNA oligonucleotides in a concentration dependent manner which correlates with the inhibition of RNA 5'-triphosphatase activity by these same oligonucleotides. We conclude that D1R1-545 represents a functionally independent domain of the mRNA capping enzyme, fully competent in substrate binding and catalysis at both the triphosphatase and guanylyltransferase active sites.
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PMID:Domain structure of the vaccinia virus mRNA capping enzyme. Expression in Escherichia coli of a subdomain possessing the RNA 5'-triphosphatase and guanylyltransferase activities and a kinetic comparison to the full-size enzyme. 866 35

The marine bioluminescent dinoflagellate Gonyaulax polyedra is capable of producing various indoleamines. The first enzyme in their formation, tryptophan hydroxylase, exhibits a high-amplitude circadian rhythm with a maximum during photophase. Hydroxyindole-O-methyltransferase shows a biphasic pattern with a major maximum during scotophase. 5-Methoxylated indoleamines, such as melatonin and 5-methoxytryptamine, peak at the beginning and in the second half of scotophase, respectively. A drop in temperature from 20 to 15 degrees C leads to dramatic increases of melatonin, up to more than 50 ng/mg protein. This effect may explain why a lower temperature sensitizes this organism to photoperiodic, indoleamine-mediated induction of asexual cysts. Melatonin can be catabolized either enzymatically or non-enzymatically. The non-enzymatic pathway involves free radicals, e.g., photooxidant cation radicals, and leads to the formation of N1-acetyl-N2- formyl-5-methoxykynuramine. Enzymatic catabolism comprises deacetylation to 5-methoxytryptamine and formation of 5-methoxytryptophol. 5-Methoxytryptamine represents a key substance acting as a stimulator of bioluminescence and a mediator of the encystment response. It opens proton channels in the membrane of an intracellular acidic vacuole system which is loaded by the action of a V-type ATPase, as shown by experiments using bafilomycin A1.
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PMID:Chronobiology of indoleamines in the dinoflagellate Gonyaulax polyedra: metabolism and effects related to circadian rhythmicity and photoperiodism. 873 41

Temperature-sensitive mutations (ts10, ts18, and ts39) of the vaccinia virus RNA helicase nucleoside triphosphate phosphohydrolase II (NPH-II) result in the production of noninfectious progeny virions at the restrictive temperature. The noninfectious mutant particles contain the wild-type complement of virion core and envelope polypeptides, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results of Western blot (immunoblot) analysis indicate that these particles lack NPH-II, whereas other enzymatic components of the virus core are present. These components include the following: DNA-dependent RNA polymerase subunits rpo147, rpo132, rpo94, rpo35, rpo30, rpo22, and rpo18; early transcription initiation factor subunits A8 and D6; mRNA capping enzyme subunits D1 and D12; RNA cap 2'-O-methyltransferase; A18 DNA helicase; DNA-dependent ATPase NPH-I; and DNA topoisomerase. Although RNA polymerase is encapsidated by the mutant viruses, mRNA synthesis in vitro by permeabilized mutant virions is only 5 to 20% that of the wild-type virus, as judged by nucleoside monophosphate incorporation into acid-insoluble material. Moreover, the transcripts synthesized by the mutant particles are longer than normal and remain virion associated. Transcription initiation by mutant virions occurs accurately at an endogenous genomic promoter, albeit at reduced levels (1 to 7%) compared with that of wild-type virions. In contrast, extracts of the mutant virions catalyze the wild-type level of transcription from an exogenous template containing an early promoter. We conclude that NPH-II is required for early mRNA synthesis uniquely in the context of the virus particle. Possible roles in transcription termination and RNA transport are discussed.
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PMID:Vaccinia virions lacking the RNA helicase nucleoside triphosphate phosphohydrolase II are defective in early transcription. 897 Sep 79


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