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PDBsum entry 1oxg
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Detection of native peptides as potent inhibitors of enzymes. Crystal structure of the complex formed between treated bovine alpha-Chymotrypsin and an autocatalytically produced fragment, Iie-Val-Asn-Gly-Glu-Glu-Ala-Val-Pro-Gly-Ser-Trp-Pro-Trp, At 2.2 angstroms resolution.
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Authors
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N.Singh,
T.Jabeen,
S.Sharma,
I.Roy,
M.N.Gupta,
S.Bilgrami,
R.K.Somvanshi,
S.Dey,
M.Perbandt,
C.Betzel,
A.Srinivasan,
T.P.Singh.
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Ref.
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FEBS J, 2005,
272,
562-572.
[DOI no: ]
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PubMed id
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Abstract
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Chymotrypsin is a prominent member of the family of serine proteases. The
present studies demonstrate the presence of a native fragment containing 14
residues from Ile16 to Trp29 in alpha-chymotrypsin that binds to chymotrypsin at
the active site with an exceptionally high affinity of 2.7 +/- 0.3 x 10(-11) M
and thus works as a highly potent competitive inhibitor. The commercially
available alpha-chymotrypsin was processed through a three phase partitioning
system (TPP). The treated enzyme showed considerably enhanced activity. The 14
residue fragment was produced by autodigestion of a TPP-treated
alpha-chymotrypsin during a long crystallization process that lasted more than
four months. The treated enzyme was purified and kept for crystallization using
vapour the diffusion method at 295 K. Twenty milligrams of lyophilized protein
were dissolved in 1 mL of 25 mM sodium acetate buffer, pH 4.8. It was
equilibrated against the same buffer containing 1.2 M ammonium sulfate. The
rectangular crystals of small dimensions of 0.24 x 0.15 x 0.10 mm(3) were
obtained. The X-ray intensity data were collected at 2.2 angstroms resolution
and the structure was refined to an R-factor of 0.192. An extra electron density
was observed at the binding site of alpha-chymotrypsin, which was readily
interpreted as a 14 residue fragment of alpha-chymotrypsin corresponding to
Ile-Val-Asn-Gly-Glu-Glu-Ala-Val-Pro-Gly-Ser-Trp-Pro-Trp(16-29). The electron
density for the eight residues of the C-terminus, i.e. Ala22-Trp29, which were
completely buried in the binding cleft of the enzyme, was of excellent quality
and all the side chains of these eight residues were clearly modeled into it.
However, the remaining six residues from the N-terminus, Ile16-Glu21 were poorly
defined although the backbone density was good. There was a continuous electron
density at 3.0 sigma between the active site Ser195 Ogamma and the carbonyl
carbon atom of Trp29 of the fragment. The final refined coordinates showed a
distance of 1.35 angstroms between Ser195 Ogamma and Trp29 C indicating the
presence of a covalent linkage between the enzyme and the native fragment. This
meant that the enzyme formed an acyl intermediate with the autodigested fragment
Ile16-Trp29. In addition to the O-C covalent bond, there were several hydrogen
bonds and hydrophobic interactions between the enzyme and the native fragment.
The fragment showed a high complementarity with the binding site of
alpha-chymotrypsin and the buried part of the fragment matched excellently with
the corresponding buried part of Turkey ovomucoid inhibitor of
alpha-chymotrypsin.
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Figure 2.
Fig. 2. The sequence of  -chymotrypsin
indicating the absence of two dipeptides as a result of
autodigestion. The segment in red from Ile16 to Trp29 is the
fragment produced by autolysis that binds to -chymotrypsin
with a high affinity.
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Figure 4.
Fig. 4. Superimposition of present complex on the complex
of chymotrypsin with the turkey ovomucoid inhibitor (red) [25]
showing similar binding mechanism of the two inhibitors. The
Pep14 fragment is shown in yellow.
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The above figures are
reprinted
by permission from the Federation of European Biochemical Societies:
FEBS J
(2005,
272,
562-572)
copyright 2005.
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