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(Received for publication, May 22, 1996, and in revised form, July 15, 1996)
From the Departments of 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
Volume 271, Number 38,
Issue of September 20, 1996
pp. 23506-23511
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
,
,
and
Biology and
¶ Chemistry, Williams College, Williamstown,
Massachusetts 01267 and the '' Department of Biochemistry and
Biophysics, Oregon State University, Corvallis, Oregon 97331
--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 µ, inhibition studies show that 8-azido-ATP acted
as a competitive inhibitor of dUTP with a Ki of
approximately 173 µ. Also, protection studies
demonstrated that nucleotide competitors inhibit photoincorporation of
the photoaffinity analogues [
-32P]5-azido-dUTP and
[
-32P]8-azido-ATP. This suggests that while catalytic
activity is limited to dUTP, other nucleotides can bind the active
site.
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