title: Purification and characterization of the vaccinia virus deoxyuridine triphosphatase expressed in Escherichia coli.
abstract: The deoxyuridine triphosphatase gene of vaccinia virus, encoded by the open reading frame F2L, was cloned into Escherichia coli and expressed under the control of a bacteriophage T7 promoter. After induction of T7 RNA polymerase by isopropyl beta-D-thiogalactopyranoside, a 16.5-kDa peptide accumulated to high levels. This 16.5-kDa protein was purified to homogeneity and characterized. Gel filtration of the purified protein revealed a trimeric native structure. Biochemical analysis revealed the enzyme to be a metalloenzyme; enzymatic activity is inhibited by EDTA. This inhibition was reversed by the addition of Mg2+, Mn2+, or Zn2+. While the enzyme activity was highly specific for dUTP with an apparent Km of 0.94 microM, inhibition studies show that 8-azido-ATP acted as a competitive inhibitor of dUTP with a Ki of approximately 173 microM. Also, protection studies demonstrated that nucleotide competitors inhibit photoincorporation of the photoaffinity analogues [gamma-32P]5-azido-dUTP and [gamma-32P]8-azido-ATP. This suggests that while catalytic activity is limited to dUTP, other nucleotides can bind the active site.
authors.affiliation: Department of Biology, Williams College, Williamstown, Massachusetts 01267, USA.
authors.forename: N A
authors.initials: NA
authors.lastname: Roseman
authors.forename: R K
authors.initials: RK
authors.lastname: Evans
authors.forename: E L
authors.initials: EL
authors.lastname: Mayer
authors.forename: M A
authors.initials: MA
authors.lastname: Rossi
authors.forename: M B
authors.initials: MB
authors.lastname: Slabaugh
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D001386:Azides / Q000494:pharmacology; D001667:Binding, Competitive; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D004791:Enzyme Inhibitors; D007700:Kinetics; D008667:Metalloproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics
chemical_list: D001386:Azides; D002413:Cations, Divalent; D003856:Deoxyuracil Nucleotides; D004791:Enzyme Inhibitors; D008667:Metalloproteins; D009711:Nucleotides; D011994:Recombinant Proteins; C027078:deoxyuridine triphosphate; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase