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Title
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Single mutation at P1 of a chymotrypsin inhibitor changes it to a trypsin inhibitor: X-ray structural (2.15 A) and biochemical basis.
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Authors
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S.Khamrui,
J.Dasgupta,
J.K.Dattagupta,
U.Sen.
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Ref.
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Biochim Biophys Acta, 2005,
1752,
65-72.
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PubMed id
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Abstract
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Change in specificity, caused by the mutations at P1 site, of the serine
protease inhibitors of different families is reported in the literature, but
Kunitz (STI) family inhibitors are almost unexplored in this regard. In this
paper, we present the crystal structure of a P1 variant of winged bean
chymotrypsin inhibitor (WCI) belonging to Kunitz (STI) family, supplemented by
biochemical, phylogenetic and docking studies on the mutant. A single mutation
(Leu-->Arg) at P1 converted WCI to a strong inhibitor of trypsin with an
association constant of 4.8x10(10) M(-1) which is comparable to other potent
trypsin inhibitors of the family. The crystal structure (2.15 A) of this mutant
(L65R) shows that its reactive site loop conformation deviates from that of WCI
and adopts a structure similar to that of Erythrina caffra trypsin inhibitor
(ETI) belonging to the same family. Mutation induced structural changes have
also been propagated in a concerted manner to the neighboring conserved
scaffolding residue Asn14, such that the side chain of this residue took an
orientation similar to that of ETI and optimized the hydrogen bonds with the
loop residues. While docking studies provide information about the accommodation
of non-specific residues in the active site groove of trypsin, the basis of the
directional alteration of the reactive site loop conformation has been
understood through sequence analysis and related phylogenetic studies.
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