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PDBsum entry 1gbt
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Hydrolase(serine proteinase)
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PDB id
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1gbt
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References listed in PDB file
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Key reference
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Title
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Structure of an acyl-Enzyme intermediate during catalysis: (guanidinobenzoyl)trypsin.
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Authors
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W.F.Mangel,
P.T.Singer,
D.M.Cyr,
T.C.Umland,
D.L.Toledo,
R.M.Stroud,
J.W.Pflugrath,
R.M.Sweet.
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Ref.
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Biochemistry, 1990,
29,
8351-8357.
[DOI no: ]
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PubMed id
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Abstract
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The crystal and molecular structure of trypsin at a transiently stable
intermediate step during catalysis has been determined by X-ray diffraction
methods. Bovine trypsin cleaved the substrate p-nitrophenyl p-guanidinobenzoate
during crystallization under conditions in which the acyl-enzyme intermediate,
(guanidinobenzoyl)trypsin, was stable. Orthorhombic crystals formed in space
group P2(1)2(1)2(1), with a = 63.74, b = 63.54, and c = 68.93 A. This is a
crystal form of bovine trypsin for which a molecular structure has not been
reported. Diffraction data were measured with a FAST (Enraf Nonius)
diffractometer. The structure was refined to a crystallographic residual of R =
0.16 for data in the resolution range 7.0-2.0 A. The refined model of
(guanidinobenzoyl)trypsin provides insight into the structural basis for its
slow rate of deacylation, which in solution at 25 degrees C and pH 7.4 exhibits
a t1/2 of 12 h. In addition to the rotation of the Ser-195 hydroxyl away from
His-157, C beta of Ser-195 moves 0.7 A toward Asp-189 at the bottom of the
active site, with respect to the native structure. This allows formation of
energetically favorable H bonds and an ion pair between the carboxylate of
Asp-189 and the guanidino group of the substrate. This movement is dictated by
the rigidity of the aromatic ring in guanidinobenzoate--model-building indicates
that this should not occur when arginine, with its more flexible aliphatic
backbone, forms the ester bond with Ser-195. As a consequence, highly ordered
water molecules in the active site are no longer close enough to the scissile
ester bond to serve as potential nucleophiles for hydrolysis.(ABSTRACT TRUNCATED
AT 250 WORDS)
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