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PDBsum entry 4rko
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Hydrolase/hydrolase inhibitor
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PDB id
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4rko
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References listed in PDB file
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Key reference
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Title
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Why ser and not thr brokers catalysis in the trypsin fold.
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Authors
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L.A.Pelc,
Z.Chen,
D.W.Gohara,
A.D.Vogt,
N.Pozzi,
E.Di cera.
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Ref.
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Biochemistry, 2015,
54,
1457-1464.
[DOI no: ]
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PubMed id
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Abstract
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Although Thr is equally represented as Ser in the human genome and as a
nucleophile is as good as Ser, it is never found in the active site of the large
family of trypsin-like proteases that utilize the Asp/His/Ser triad. The
molecular basis of the preference of Ser over Thr in the trypsin fold was
investigated with X-ray structures of the thrombin mutant S195T free and bound
to an irreversible active site inhibitor. In the free form, the methyl group of
T195 is oriented toward the incoming substrate in a conformation seemingly
incompatible with productive binding. In the bound form, the side chain of T195
is reoriented for efficient substrate acylation without causing steric clash
within the active site. Rapid kinetics prove that this change is due to
selection of an active conformation from a preexisting ensemble of reactive and
unreactive rotamers whose relative distribution determines the level of activity
of the protease. Consistent with these observations, the S195T substitution is
associated with a weak yet finite activity that allows identification of an
unanticipated important role for S195 as the end point of allosteric
transduction in the trypsin fold. The S195T mutation abrogates the
Na(+)-dependent enhancement of catalytic activity in thrombin, activated protein
C, and factor Xa and significantly weakens the physiologically important
allosteric effects of thrombomodulin on thrombin and of cofactor Va on factor
Xa. The evolutionary selection of Ser over Thr in trypsin-like proteases was
therefore driven by the need for high catalytic activity and efficient
allosteric regulation.
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Secondary reference #1
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Title
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Molecular dissection of na+ binding to thrombin.
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Authors
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A.O.Pineda,
C.J.Carrell,
L.A.Bush,
S.Prasad,
S.Caccia,
Z.W.Chen,
F.S.Mathews,
E.Di cera.
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Ref.
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J Biol Chem, 2004,
279,
31842-31853.
[DOI no: ]
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PubMed id
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Figure 7.
FIG. 7. Stereo view of the Na^+ binding environment in the
structures of F (free fast form, gold), S (free slow form, red),
FL (PPACK-bound fast form, blue), and SL (PPACK-bound slow form,
green). Shown are all atoms within 3 Å of the bound Na^+
in the F structure, in addition to the side chains of Asp-189
and Asp-221. Note the similarity of the Na^+ coordination shell
between F and FL; the bound Na^+ is coordinated octahedrally by
the backbone O atoms of Lys-224 and Arg-221a and by four buried
water molecules that H-bond to (clockwise) Asp-189, Asp-221,
Gly-223, and Tyr-184a. Only some of these water molecules are
replaced in the absence of Na^+ (S and SL). Note the
rearrangement of the side chain of Asp-189 in the S structure
and the significant shift in the backbone O atom of Arg-221a
that assumes a position incompatible with Na^+ coordination.
H-bonds are shown by broken lines and refer to the F structure.
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Figure 8.
FIG. 8. Stereo view of the electron density maps of the S
(A), F (B), SL (C), and FL (D) intermediates of thrombin in the
regions bearing the most significant structural transitions.
Residues are rendered in CPK. The bound Na^+ is rendered as a
cyan ball. Shown are the 221–224 loop region and the 187–195
domain. Note how Asp-222 and Arg-187 have joined densities in
the F form, indicative of ion pair interaction, but not in the S
form. Also notable are the reorientation of Asp-189 and Glu-192
in the S form, as well as the shift in the position of Ser-195.
Other changes observed in the slow  fast transition involve
the network of water molecules (red balls) embedding the Na^+
site, the S1 pocket, and the active site region. In the fast
form, this network is well organized and contains 11 water
molecules. In the slow form, the water molecules are reduced to
seven, and the long range connectivity of the network is lost
(see also Fig. 9). The 2F[o] - F[c] electron density maps are
contoured at 0.7  for S and F and at 1.0
for
SL and FL.
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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