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PDBsum entry 1thp
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Hydrolase/hydrolase inhibitor
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PDB id
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1thp
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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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Unexpected crucial role of residue 225 in serine proteases.
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Authors
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E.R.Guinto,
S.Caccia,
T.Rose,
K.Fütterer,
G.Waksman,
E.Di cera.
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Ref.
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Proc Natl Acad Sci U S A, 1999,
96,
1852-1857.
[DOI no: ]
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PubMed id
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Abstract
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Residue 225 in serine proteases of the chymotrypsin family is Pro or Tyr in more
than 95% of nearly 300 available sequences. Proteases with Y225 (like some blood
coagulation and complement factors) are almost exclusively found in vertebrates,
whereas proteases with P225 (like degradative enzymes) are present from bacteria
to human. Saturation mutagenesis of Y225 in thrombin shows that residue 225
affects ligand recognition up to 60,000-fold. With the exception of Tyr and Phe,
all residues are associated with comparable or greatly reduced catalytic
activity relative to Pro. The crystal structures of three mutants that differ
widely in catalytic activity (Y225F, Y225P, and Y225I) show that although
residue 225 makes no contact with substrate, it drastically influences the shape
of the water channel around the primary specificity site. The activity profiles
obtained for thrombin also suggest that the conversion of Pro to Tyr or Phe
documented in the vertebrates occurred through Ser and was driven by a
significant gain (up to 50-fold) in catalytic activity. In fact, Ser and Phe are
documented in 4% of serine proteases, which together with Pro and Tyr account
for almost the entire distribution of residues at position 225. The unexpected
crucial role of residue 225 in serine proteases explains the evolutionary
selection of residues at this position and shows that the structural
determinants of protease activity and specificity are more complex than
currently believed. These findings have broad implications in the rational
design of enzymes with enhanced catalytic properties.
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Figure 1.
Fig. 1. Effect of residue 225 on the Na^+ specificity of
thrombin, measured as the ratio of the k[cat]/K[m] values for
the hydrolysis of H-D-Phe-Pro-Arg-p-nitroanilide (FPR) in the
presence of 200 mM NaCl or choline chloride, 5 mM Tris, 0.1%
polyethylene glycol, pH 8.0 at 25°C (6). Binding of Na^+ to
wild type (Tyr) enhances k[cat]/K[m] nearly 25-fold. A
significant enhancement also is observed for Phe, His, Met, Gln,
Trp, and Ser, in decreasing order. All other residues show no
significant difference between Na^+ and choline.
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Figure 3.
Fig. 3. Effect of residue 225 on the architecture of the
water channel around the primary specificity site of thrombin.
Shown is a cross section of the enzyme along the water channel
that reveals the active site inhibitor PPACK (purple), D189 in
the primary specificity site, residue 225 with the carbonyl O
atom of residue 224 (red), buried water molecules (blue), and
Na^+ (yellow). The surface of the enzyme is rendered as a net
(black, above the plan of section; green, below it). The side
chain of residue 225 points away from D189 and makes no contact
with PPACK. The Y225F mutant is practically identical to wild
type (11) and shows a bound Na^+ coordinated octahedrally by the
carbonyl O atoms of K224 and R221a (not shown), and four water
molecules (23, 26). In this mutant, the water channel connects
the active site to an aperture at the bottom of the molecule
(arrow). In the Y225P mutant, there is no evidence of bound
Na^+; the carbonyl O atom of K224 is shifted 70° toward the
interior of the channel and occludes it in the middle. In
addition, the channel is shunted laterally (arrow) around
residue 225 because of the Y225P replacement. In the Y225I
mutant, the channel has three apertures.
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