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PDBsum entry 2ra3
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Hydrolase/hydrolase inhibitor
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PDB id
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2ra3
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References listed in PDB file
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Key reference
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Title
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Structural basis for accelerated cleavage of bovine pancreatic trypsin inhibitor (bpti) by human mesotrypsin.
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Authors
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M.A.Salameh,
A.S.Soares,
A.Hockla,
E.S.Radisky.
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Ref.
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J Biol Chem, 2007,
283,
4115-4123.
[DOI no: ]
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PubMed id
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Abstract
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Human mesotrypsin is an isoform of trypsin that displays unusual resistance to
polypeptide trypsin inhibitors, and has been observed to cleave several such
inhibitors as substrates. While substitution of arginine for the highly
conserved glycine-193 in the trypsin active site has been implicated as a
critical factor in the inhibitor resistance of mesotrypsin, how this
substitution leads to accelerated inhibitor cleavage is not clear. Bovine
pancreatic trypsin inhibitor (BPTI) forms an extremely stable and
cleavage-resistant complex with trypsin, and thus provides a rigorous challenge
of mesotrypsin catalytic activity toward polypeptide inhibitors. Here, we report
kinetic constants for mesotrypsin and the highly homologous (but inhibitor
sensitive) human cationic trypsin, describing inhibition by and cleavage of
BPTI, as well as crystal structures of the mesotrypsin-BPTI and human cationic
trypsin-BPTI complexes. We find that mesotrypsin cleaves BPTI with a rate
constant accelerated 350-fold over that of human cationic trypsin and
150,000-fold over that of bovine trypsin. From the crystal structures, we see
that small conformational adjustments limited to several side chains enable
mesotrypsin-BPTI complex formation, surmounting the predicted steric clash
introduced by Arg-193. Our results show that the mesotrypsin-BPTI interface
favors catalysis through (a) electrostatic repulsion between the closely spaced
mesotrypsin Arg-193 and BPTI Arg-17, and (b) elimination of two hydrogen bonds
between the enzyme and the amine leaving group portion of BPTI. Our model
predicts that these deleterious interactions accelerate leaving group
dissociation and deacylation.
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Figure 3.
FIGURE 3. Mesotrypsin conformational changes upon BPTI
binding. A, superposition of the mesotrypsin-BPTI complex
(mesotrypsin in orange and BPTI in blue/cyan) with the
mesotrypsin-benzamidine complex (mesotrypsin in beige and
benzamidine in red) highlights several differences induced by
BPTI binding. Significant movements in the positions of side
chains, illustrated by the black arrows, include the upward
displacement of Arg-193 by  6 Å displacing a
water molecule, adoption of an alternate rotamer by His-40, and
displacement of Asp-153 by 1 Å to enable formation of a
water-bridged ionic interaction with Arg-193. B, environment
surrounding the BPTI-bound position of mesotrypsin Arg-193 is
shown with 2F[o]-F[c] map contoured at 1.5 sigma. Arg-193 forms
direct H-bonds to the carbonyl oxygens of Trp-141 and Pro-152,
and water bridged interactions with the side chains of Asp-153
and BPTI Arg-17.
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Figure 4.
FIGURE 4. Differences between the mesotrypsin-BPTI and
human cationic trypsin-BPTI interfaces. In this superposition of
the two complexes, mesotrypsin and BPTI are colored orange and
blue/cyan as in the previous figure, while cationic trypsin and
its bound BPTI are colored dark and light gray, respectively.
Three H-bonds present only in the cationic trypsin complex (see
text) are shown as dotted lines.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2007,
283,
4115-4123)
copyright 2007.
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