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Title
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cDNA-derived sequence of UMP-CMP kinase from Dictyostelium discoideum and expression of the enzyme in Escherichia coli.
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Authors
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L.Wiesmüller,
A.A.Noegel,
O.Bârzu,
G.Gerisch,
M.Schleicher.
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Ref.
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J Biol Chem, 1990,
265,
6339-6345.
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PubMed id
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Abstract
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A cDNA coding for UMP-CMP kinase from Dictyostelium discoideum was isolated from
a lambda gt11 expression library and sequenced. The corresponding mRNA has a
size of 0.7 kilobase and is down-regulated during early development of D.
discoideum. Southern blotting demonstrated that the UMP-CMP kinase is encoded by
a single gene. The deduced amino acid sequence of UMP-CMP kinase shows a high
degree of homology with adenylate kinases from different sources with the
highest degree of homology to cytosolic adenylate kinase from vertebrate muscle
(43%). The enzyme expressed in Escherichia coli after cloning the cDNA into an
ATG expression vector was purified and analyzed for its structural and kinetic
properties. The UMP-CMP kinase uses preferentially ATP (Km,app = 25 microM) as
phosphate donor and is specific for UMP (Km,app = 0.4 mM) and CMP (Km,app = 0.1
mM). The enzyme is strongly inhibited by the substrate analogue
P1-(adenosine-5')-P5-(uridine-5')-pentaphosphate (Ki between 0.05 and 0.1
microM) and is inactivated by modification of free thiol groups with
5,5'-dithiobis(2-nitrobenzoic acid).
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