Purification and characterization of the vaccinia virus deoxyuridine triphosphatase expressed in Escherichia coli.
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.
Roseman
N A
NA
Department of Biology, Williams College, Williamstown, Massachusetts 01267, USA.
Evans
R K
RK
Mayer
E L
EL
Rossi
M A
MA
Slabaugh
M B
MB
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
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