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Title
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Purification, catalytic properties, and thermal stability of threo-Ds-3-isopropylmalate dehydrogenase coded by leuB gene from an extreme thermophile, Thermus thermophilus strain HB8.
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Authors
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T.Yamada,
N.Akutsu,
K.Miyazaki,
K.Kakinuma,
M.Yoshida,
T.Oshima.
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Ref.
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J Biochem (tokyo), 1990,
108,
449-456.
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PubMed id
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Abstract
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Threo-Ds-3-isopropylmalate dehydrogenase coded by the leuB gene from an extreme
thermophile, Thermus thermophilus strain HB8, was expressed in Escherichia coli
carrying a recombinant plasmid. The thermostable enzyme thus produced was
extracted from the E. coli cells, purified, and crystallized. The enzyme was
shown to be a dimer of identical subunits of molecular weight (4.0 +/- 0.5) x
10(4). The Km for threo-Ds-3-isopropylmalate was estimated to be 8.0 x 10(-5) M
and that for NAD 6.3 x 10(-4) M. The optimum pH at 75 degrees C in the presence
of 1.2 M KCl was around 7.2. The presence of Mg2+ or Mn2+ was essential for the
enzyme action. The enzyme was activated about 30-fold by the addition of 1 M KCl
or RbCl. The high salt concentration decelerated the thermal unfolding of the
enzyme, and accelerated the aggregation of the unfolded protein. Based on these
effects, the molecular mechanism of the unusual stability of the enzyme is
discussed.
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