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:3.6.1.25 (triphosphatase)
1,529 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

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

D1R1-545, an active subdomain of the large subunit of vaccinia virus mRNA capping enzyme possessing ATPase, RNA 5'-triphosphatase, and guanylyltransferase activities, was expressed in Escherichia coli and shown to be functionally equivalent to the heterodimeric enzyme (Myette, J. R., and Niles, E. G. (1996) J. Biol. Chem. 271, 11936-11944). A detailed characterization of the phosphohydrolytic activities of D1R1-545 demonstrates that, in addition to ATPase and RNA 5'-triphosphatase activities, the capping enzyme also possesses a general nucleoside triphosphate phosphohydrolase activity that lacks a preference for the nucleoside base or sugar. Nucleoside triphosphate and mRNA saturation kinetics are markedly different, with RNA exhibiting a Km and turnover number 100- and 10-fold less, respectively, than those values measured for any NTP. The linear competitive inhibition of RNA 5'-triphosphatase activity by ATP, and the relative manner by which both ATPase and RNA 5'-triphosphatase activities are inhibited by specific oligonucleotides, kinetically demonstrate that each activity is carried out at a common active site. Direct UV photo-cross-linking of either 32P-radiolabeled ATP or 23-mer triphosphorylated RNA, followed by cyanogen bromide cleavage of the photo-linked enzyme, localizes the major binding site for both ATP and RNA to a region between amino acids 1 and 221. The inability of ATP to competitively inhibit either E approximately GMP formation or the transfer of GMP to RNA kinetically differentiates the phosphohydrolase active site from the guanylyltransferase active site.
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PMID:Characterization of the vaccinia virus RNA 5'-triphosphatase and nucleotide triphosphate phosphohydrolase activities. Demonstrate that both activities are carried out at the same active site. 866 36

Vaccinia virus mRNA capping enzyme is a multifunctional protein with RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7-) methyltransferase activities. The enzyme is a heterodimer of 95- and 33-kDa subunits encoded by the vaccinia virus D1 and D12 genes, respectively. The N-terminal 60-kDa of the D1 subunit (from residues 1 to 545) is an autonomous domain which catalyzes the triphosphatase and guanylyltransferase reactions. Mutations in the D1 subunit that specifically inactivate the guanylyltransferase without affecting the triphosphatase component have been described (P. Cong and S. Shuman, Mol. Cell. Biol. 15:6222-6231, 1995). In the present study, we identified two alanine-cluster mutations of D1(1-545), R77A-K79A and E192A-E194A, that selectively inactivated the triphosphatase, but not the guanylyltransferase. Concordant mutational inactivation of RNA triphosphatase and nucleoside triphosphatase functions (to approximately 1% of wild-type specific activity) suggests that both gamma-phosphate cleavage reactions occur at a single active site. The R77A-K79A and E192A-E194A mutant enzymes were less active than wild-type D1(1-545) in the capping of triphosphate-terminated poly(A) but could be complemented in vitro by D1(1-545)-K260A, which is inert in nucleotidyl transfer but active in gamma-phosphate cleavage. Whereas wild-type D1(1-545) formed only the standard GpppA cap, the R77A-K79A and E192A-E194A enzymes synthesized an additional dinucleotide, GppppA. This finding illuminates a novel property of the vaccinia virus capping enzyme, the use of triphosphate RNA ends as an acceptor for nucleotidyl transfer when gamma-phosphate cleavage is rate limiting.
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PMID:Mutational analysis of the RNA triphosphatase component of vaccinia virus mRNA capping enzyme. 870 42

The yeast mRNA capping enzyme is composed of 52 (alpha) and 80 kDa (beta) polypeptides, which are responsible for its mRNA guanylyltransferase and RNA 5'-triphosphatase activities, respectively. We isolated the gene encoding the alpha subunit (CEG1) and showed that CEG1 is essential for yeast cell growth [Shibagaki et al., (1992) J. Biol. Chem. 267, 9521-9528]. In this study, CEG1 was expressed in Escherichia coli and the alpha subunit protein was purified to near homogeneity. A [32P]GMP-bound tryptic peptide derived from the recombinant enzyme-[32P]GMP covalent reaction intermediate was converted to a [32P]phosphoryl-peptide through periodate oxidation followed by beta-elimination. Hydrolysis of the [32P]phosphoryl-peptide with alkali resulted in [32P]N epsilon-phospholysine as the only phosphoamino acid, indicating that GMP in the enzyme-GMP complex is bound to a lysine residue via a phosphoamide linkage. Microsequencing of the [32P]GMP-peptide showed that the GMP binding site was located in the region between amino acids 60 and 75, which contained an internal trypsin-resistant lysine at position 70. CEG1 was subjected to site-directed mutagenesis and the mutant proteins were expressed in E. coli. Substitution of His or Ile for Lys70 entirely abolished the enzyme-GMP formation activity, and this mutation was lethal to yeast in vivo, supporting the notion that the active site in the alpha subunit is located at Lys70. Replacement of Lys70 with Arg reduced the ability to form the enzyme-GMP complex; however, yeast cells bearing this allele were not viable. A series of mutations, including 8 amino acid replacements and 3 insertions, near the active site (Lys70-Thr-Asp-Gly motif) were also introduced and the mutant polypeptides were examined for catalytic activity in vitro as well as yeast cell viability in vivo. There was a good correlation between the in vitro and in vivo functions of the mutant proteins, except when Asp72 was replaced with Glu, which allowed formation of the enzyme-GMP complex but failed to support cell growth. The results with Lys70 to Arg and Asp72 to Glu substitutions indicated that guanylyltransfer to RNA and/or additional roles besides cap formation per se are impaired in these mutant proteins.
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PMID:Localization and in vitro mutagenesis of the active site in the Saccharomyces cerevisiae mRNA capping enzyme. 872 Jan 51

The yeast Saccharomyces cerevisiae mRNA capping enzyme is composed of two subunits of alpha (52 kDa, mRNA guanylyltransferase) and beta (80 kDa, RNA 5'-triphosphatase). We have isolated the alpha subunit gene (CEG1) by immunological screening. In this report, with the aid of partial amino acid sequences of purified yeast capping enzyme, we isolated the gene, designated CET1, encoding the S. cerevisiae capping enzyme beta subunit. Amino acid sequence analysis revealed that the gene encodes for 549 amino acids with a calculated M(r) of 61,800 which is unexpectedly smaller than the size estimated by SDS-PAGE. Gene disruption experiment showed that CET1 is essential for yeast cell growth. The purified recombinant CET1 gene product, Cet1, exhibited an RNA 5'-triphosphatase activity which specifically removed the gamma-phosphate from the triphosphate-terminated RNA substrate, but not from nucleoside triphosphates, confirming the identity of the gene. Interaction between the Cet1 and the Ceg1 was also studied by the West-Western procedure using recombinant Ceg1-[32P]GMP as probe.
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PMID:Isolation and characterization of the yeast mRNA capping enzyme beta subunit gene encoding RNA 5'-triphosphatase, which is essential for cell viability. 934 80

The N-terminal 60 kDa (amino acids 1 to 545) of the D1 subunit of vaccinia virus mRNA capping enzyme is an autonomous bifunctional domain with triphosphatase and guanylyltransferase activities. We previously described two alanine cluster mutations, R77 to A (R77A)-K79A and E192A-E194A, which selectively inactivated the triphosphatase component. Here, we characterize the activities of 11 single alanine mutants-E37A, E39A, Q60A, E61A, T67A, T69A, K75A, R77A, K79A, E192A, and E194A-and a quadruple mutant in which four residues (R77, K79, E192, and E194) were replaced by alanine. We report that Glu-37, Glu-39, Arg-77, Glu-192, and Glu-194 are essential for gamma-phosphate cleavage. The five essential residues are conserved in the capping enzymes of Shope fibroma virus, molluscum contagiosum virus, and African swine fever virus. Probing the structure of D1(1-545) by limited V8 proteolysis suggested a bipartite subdomain structure. The essential residue Glu-192 is the principal site of V8 cleavage. Secondary cleavage by V8 occurs at the essential residue Glu-39. The triphosphatase-defective quadruple mutant transferred GMP to the triphosphate end of poly(A) to form a tetraphosphate cap structure, GppppA. We report that GppppA-capped RNA is a poor substrate for cap methylation by the vaccinia virus and Saccharomyces cerevisiae RNA (guanine-7) methyltransferases. The transcription termination factor activity of the D1-D12 capping enzyme heterodimer was not affected by mutations that abrogated ATPase activity. Thus, the capping enzyme is not responsible for the requirement for ATP hydrolysis during transcription termination.
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PMID:Structure-function analysis of the triphosphatase component of vaccinia virus mRNA capping enzyme. 937 57

5'-Capping is an early mRNA modification that has important consequences for downstream events in gene expression. We have isolated mammalian cDNAs encoding capping enzyme. They contain the sequence motifs characteristic of the nucleotidyl transferase superfamily. The predicted mouse and human enzymes consist of 597 amino acids and are 95% identical. Mouse cDNA directed synthesis of a guanylylated 68-kDa polypeptide that also contained RNA 5'-triphosphatase activity and catalyzed formation of RNA 5'-terminal GpppG. A haploid strain of Saccharomyces cerevisiae lacking mRNA guanylyltransferase was complemented for growth by the mouse cDNA. Conversion of Lys-294 in the KXDG-conserved motif eliminated both guanylylation and complementation, identifying it as the active site. The K294A mutant retained RNA 5'-triphosphatase activity, which was eliminated by N-terminal truncation. Full-length capping enzyme and an active C-terminal fragment bound to the elongating form and not to the initiating form of polymerase. The results document functional conservation of eukaryotic mRNA guanylyltransferases from yeast to mammals and indicate that the phosphorylated C-terminal domain of RNA polymerase II couples capping to transcription elongation. These results also explain the selective capping of RNA polymerase II transcripts.
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PMID:Mammalian capping enzyme complements mutant Saccharomyces cerevisiae lacking mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase II. 939 72

Previous studies demonstrated that the mammalian mRNA capping enzyme is a bifunctional enzyme containing RNA 5'-triphosphatase and mRNA guanylyl-transferase activities in a single polypeptide. In yeast, both the above activities are separated into two different subunits, alpha and beta, the genes for which we have cloned recently. It is thus interesting to compare the structural and functional relationships between the mammalian and yeast capping enzymes. Here we isolated two human cDNAs encoding mRNA capping enzymes termed hCAP1a and hCAP1b which encode 597 and 541 amino acids, respectively. They are different only at the region coding for the C-terminal portion of the enzyme. Comparison of the deduced amino acid sequences with other cellular and viral capping enzymes showed that all the regions conserved among mRNA guanylyltransferases are observed in our clones except one conserved C-terminal region which was absent in the hCAP1b protein. The purified recombinant hCAP1a gene product, hCAP1a, exhibited both RNA 5'-triphosphatase and mRNA guanylyltransferase activities. Deletion mutant analysis of hCAP1a showed that the N-terminal 213 amino acid fragment containing a tyrosine specific protein phosphatase motif catalyzed the RNA 5'-triphosphatase activity and the C-terminal 369 amino acid fragment exhibited the mRNA guanylyltransferase activity. On the other hand, hCAP1b showed RNA 5'-triphosphatase activity, but neither enzyme-GMP covalent complex formation nor cap structure formation was detected.
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PMID:Cloning and characterization of two human cDNAs encoding the mRNA capping enzyme. 947 87

The human mRNA 5'-capping enzyme cDNA was identified. Three highly related cDNAs, HCE1 (human mRNAcappingenzyme1), HCE1A and HCE1B , were isolated from a HeLa cDNA library. The HCE1 cDNA has the longest ORF, which can encode a 69 kDa protein. A short region of 69 bp in the 3'-half of the HCE1 ORF was missing in HCE1A and HCE1B , and, additionally, HCE1B has an early translation termination signal, which suggests that the latter two cDNAs represent alternatively spliced product. When expressed in Escherichia coli as a fusion protein with glutathione S -transferase, Hce1p displayed both mRNA 5'-triphosphatase (TPase) and mRNA 5'-guanylyltransferase (GTase) activities, and it formed a cap structure at the 5'-triphosphate end of RNA, demonstrating that it indeed specifies an active mRNA 5'-capping enzyme. The recombinant proteins derived from HCE1A and HCE1B possessed only TPase activity. When expressed from ADH1 promoter, HCE1 but not HCE1A and HCE1B complemented Saccharomyces cerevisiae CEG1 and CET1 , the genes for GTase and TPase, respectively. These results demonstrate that the N-terminal part of Hce1p is responsible for TPase activity and the C-terminal part is essential for GTase activity. In addition, the human TPase domain cannot functionally substitute for the yeast enzyme in vivo.
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PMID:Isolation and characterization of a human cDNA for mRNA 5'-capping enzyme. 951 41


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