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PDBsum entry 1fy8
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Hydrolase/hydrolase inhibitor
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PDB id
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1fy8
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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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The energetic cost of induced fit catalysis: crystal structures of trypsinogen mutants with enhanced activity and inhibitor affinity.
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Authors
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A.Pasternak,
A.White,
C.J.Jeffery,
N.Medina,
M.Cahoon,
D.Ringe,
L.Hedstrom.
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Ref.
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Protein Sci, 2001,
10,
1331-1342.
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PubMed id
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Abstract
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The contribution of induced fit to enzyme specificity has been much debated,
although with little experimental data. Here we probe the effect of induced fit
on enzyme specificity using the trypsin(ogen) system. BPTI is known to induce
trypsinogen to assume a trypsinlike conformation. Correlations are observed
between BPTI affinity and the values of k(cat)/K(m) for the hydrolysis of two
substrates by eight trypsin(ogen) variants. The slope of both correlations is
-1.8. The crystal structures of the BPTI complexes of four variant trypsinogens
were also solved. Three of these enzymes, K15A, DeltaI16V17/D194N, and
DeltaI16V17/Q156K trypsinogen, are 10- to 100-fold more active than trypsinogen.
The fourth variant, DeltaI16V17 trypsinogen, is the lone outlier in the
correlations; its activity is lower than expected based on its affinity for
BPTI. The S1 site and oxyanion hole, formed by segments 184A-194 and 216-223,
are trypsinlike in all of the enzymes. These structural and kinetic data confirm
that BPTI induces an active conformation in the trypsin(ogen) variants. Thus,
changes in BPTI affinity monitor changes in the energetic cost of inducing a
trypsinlike conformation. Although the S1 site and oxyanion hole are similar in
all four variants, the N-terminal and autolysis loop (residues 142-152) segments
have different interactions for each variant. These results indicate that
zymogen activity is controlled by a simple conformational equilibrium between
active and inactive conformations, and that the autolysis loop and N-terminal
segments control this equilibrium. Together, these data illustrate that induced
fit does not generally contribute to enzyme specificity.
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