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Title
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Role of Calcium in the thermal stability of thermolysin.
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Authors
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F.W.Dahlquist,
J.W.Long,
W.L.Bigbee.
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Ref.
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Biochemistry, 1976,
15,
1103-1111.
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PubMed id
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Abstract
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The effect of calcium ion on the thermal stability of thermolysin has been
investigated. The native protein undergoes an irreversible structural change and
autolysis at high temperature. Analysis of the calcium ion dependence of the
apparent melting temperature observed spectroscopically gives an apparent deltaH
of -x (130 kcal/mol) where x is either 1 or 2. Neither zinc ion, where bound at
the active site, nor terbium ion, which binds very tightly to the double calcium
binding site, shows a stabilizing effect. These sites are therefore presumably
not coupled to the transition which leads to autolysis. Removal of calcium ion
from the native enzyme at temperatures below 50 degrees C results in
inactivation but not major autolysis. The addition of 1 equiv of terbium before
calcium removal results in a protein species which is 40% active and is no
longer subject to thermal stabilization by calcium. These results suggest a
pathway for the thermal inactivation of the enzyme which involves an
irreversible structural change at one or both of the single calcium ion sites.
This change propagates to the active site and results in inactivation. The
binding of calcium ion to either or both single sites completely inhibits this
structural change. The structural change is apparently cooperative and may
correspond to a localized denaturation of the native structure.
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