 |
PDBsum entry 1z8i
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Hydrolase/hydrolase inhibitor
|
PDB id
|
|
|
|
1z8i
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
Contents |
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
* Residue conservation analysis
|
|
|
|
|
|
|
|
|
|
Enzyme class:
|
|
Chains A, B:
E.C.3.4.21.5
- thrombin.
|
|
|
|
|
|
|
Reaction:
|
|
Preferential cleavage: Arg-|-Gly; activates fibrinogen to fibrin and releases fibrinopeptide A and B.
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
|
|
|
|
|
|
| |
|
DOI no:
|
J Biol Chem
280:25644-25650
(2005)
|
|
PubMed id:
|
|
|
|
|
|
| |
|
Energetic and structural consequences of perturbing Gly-193 in the oxyanion hole of serine proteases.
|
|
K.M.Bobofchak,
A.O.Pineda,
F.S.Mathews,
E.Di Cera.
|
|
|
|
|
| |
ABSTRACT
|
|
|
|
| |
|
|
The oxyanion hole of serine proteases is formed by the backbone N atoms of the
catalytic Ser-195 and Gly-193 and engages the backbone O atom of the P1 residue
of substrate in an important H-bonding interaction. The energetic contribution
of this interaction in the ground and transition states is presently unknown.
Measurements of the individual rate constants defining the catalytic mechanism
of substrate hydrolysis for wild-type thrombin and trypsin and their G193A and
G193P mutants reveal that Gly-193 is required for optimal substrate binding and
acylation. Crystal structures of the G193A and G193P mutants of thrombin bound
to the active site inhibitor H-d-Phe-Pro-Arg-CH2Cl document the extent of
perturbation induced by the replacement of Gly-193. The Ala mutant weakens the
H-bonding interaction of the N atom of residue 193, whereas the Pro substitution
abrogates it altogether with additional small shifts of the protein backbone.
From the kinetic and structural data, we estimate that the H-bonding interaction
in the oxyanion hole contributes a stabilization of the ground and transition
states of > 1.5 kcal/mol but < 3.0 kcal/mol. These results shed light on a
basic aspect of the enzyme-substrate interaction in the entire family of
trypsin-like serine proteases.
|
|
|
|
|
|
| |
Selected figure(s)
|
|
|
|
| |
|
|
|
|
|
|
Figure 1.
FIG. 1. Arrhenius plots of the specificity constant s =
k[cat]/K[m] and k[cat] for the cleavage of FPR by thrombin (A)
and trypsin (B) wild-type (  ) and mutants G193A (
 ) and
G193P ( o ), in the temperature range from 5 °C to 45
°C. Note the use of the decimal logarithm in the ordinate.
Experimental conditions are as follows: 5 mM Tris, 0.1%
polyethylene glycol, 200 mM NaCl, pH 8.0. Continuous lines were
drawn according to Eqs. 4 and 5 in the text, with best fit
parameter values listed in Table II.
|
|
Figure 2.
FIG. 2. Stereoview of the molecular architecture of the
H-bonding (dashed lines) network of interactions between
thrombin and PPACK. Shown is the overlay of wild-type thrombin
in the FL form (18) (yellow) with the mutants G193A (blue; shown
with H-bonds) and G913P (red). The distances of these
interactions are listed in Table III. The position of the N atom
of residue 193 in the G193A and G193P mutants is indicated by an
arrow.
|
|
|
|
|
|
| |
The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2005,
280,
25644-25650)
copyright 2005.
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
 |
 |
|
Literature references that cite this PDB file's key reference
|
|
|
| |
PubMed id
|
|
Reference
|
|
|
|
|
|
E.Di Cera
(2009).
Serine proteases.
|
| |
IUBMB Life,
61,
510-515.
|
|
|
|
|
|
|
W.Niu,
Z.Chen,
L.A.Bush-Pelc,
A.Bah,
P.S.Gandhi,
and
E.Di Cera
(2009).
Mutant N143P reveals how Na+ activates thrombin.
|
| |
J Biol Chem,
284,
36175-36185.
|
|
|
PDB codes:
|
|
|
|
|
|
|
|
A.E.Schmidt,
M.F.Sun,
T.Ogawa,
S.P.Bajaj,
and
D.Gailani
(2008).
Functional role of residue 193 (chymotrypsin numbering) in serine proteases: influence of side chain length and beta-branching on the catalytic activity of blood coagulation factor XIa.
|
| |
Biochemistry,
47,
1326-1335.
|
|
|
|
|
|
|
A.Korostelev,
H.Asahara,
L.Lancaster,
M.Laurberg,
A.Hirschi,
J.Zhu,
S.Trakhanov,
W.G.Scott,
and
H.F.Noller
(2008).
Crystal structure of a translation termination complex formed with release factor RF2.
|
| |
Proc Natl Acad Sci U S A,
105,
19684-19689.
|
|
|
PDB codes:
|
|
|
|
|
|
|
|
E.Di Cera
(2008).
Thrombin.
|
| |
Mol Aspects Med,
29,
203-254.
|
|
|
|
|
|
|
N.L.Zakharchenko,
E.A.Ermakova,
and
I.u.F.Zuev
(2008).
[Effect of trypsin microenvironment on the rate constants of elementary stages of Nalpha-benzoyl-L-arginine ethyl ester hydrolysis]
|
| |
Bioorg Khim,
34,
404-408.
|
|
|
|
|
|
|
D.Gailani,
A.Schmidt,
M.F.Sun,
P.H.Bolton-Maggs,
and
S.P.Bajaj
(2007).
A cross-reactive material positive variant of coagulation factor XI (FXIP520L) with a catalytic defect.
|
| |
J Thromb Haemost,
5,
781-787.
|
|
|
|
|
|
|
J.Tóth,
Z.Simon,
P.Medveczky,
L.Gombos,
B.Jelinek,
L.Szilágyi,
L.Gráf,
and
A.Málnási-Csizmadia
(2007).
Site directed mutagenesis at position 193 of human trypsin 4 alters the rate of conformational change during activation: role of local internal viscosity in protein dynamics.
|
| |
Proteins,
67,
1119-1127.
|
|
|
|
|
|
|
E.Szepessy,
and
M.Sahin-Tóth
(2006).
Human mesotrypsin exhibits restricted S1' subsite specificity with a strong preference for small polar side chains.
|
| |
FEBS J,
273,
2942-2954.
|
|
|
|
|
|
The most recent references are shown first.
Citation data come partly from CiteXplore and partly
from an automated harvesting procedure. Note that this is likely to be
only a partial list as not all journals are covered by
either method. However, we are continually building up the citation data
so more and more references will be included with time.
Where a reference describes a PDB structure, the PDB
codes are
shown on the right.
|
|
|
Links
