|
|
|
|
|
 |
Contents |
 |
|
|
|
|
|
|
|
|
|
|
|
|
* Residue conservation analysis
|
|
|
|
|
|
|
|
|
|
Enzyme class:
|
|
Chains B, C:
E.C.3.4.21.1
- chymotrypsin.
|
|
|
|
|
|
|
Reaction:
|
|
Preferential cleavage: Tyr-|-Xaa, Trp-|-Xaa, Phe-|-Xaa, Leu-|-Xaa.
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
|
|
|
|
|
|
| |
|
DOI no:
|
Biochemistry
40:2439-2447
(2001)
|
|
PubMed id:
|
|
|
|
|
|
| |
|
Correlation of low-barrier hydrogen bonding and oxyanion binding in transition state analogue complexes of chymotrypsin.
|
|
D.Neidhart,
Y.Wei,
C.Cassidy,
J.Lin,
W.W.Cleland,
P.A.Frey.
|
|
|
|
|
| |
ABSTRACT
|
|
|
|
| |
|
|
The structures of the hemiketal adducts of Ser 195 in chymotrypsin with
N-acetyl-L-leucyl-L-phenylalanyl trifluoromethyl ketone (AcLF-CF3) and
N-acetyl-L-phenylalanyl trifluoromethyl ketone (AcF-CF3) were determined to
1.4-1.5 A by X-ray crystallography. The structures confirm those previously
reported at 1.8-2.1 A [Brady, K., Wei, A., Ringe, D., and Abeles, R. H. (1990)
Biochemistry 29, 7600-7607]. The 2.6 A spacings between Ndelta1 of His 57 and
Odelta1 of Asp 102 are confirmed at 1.3 A resolution, consistent with the
low-barrier hydrogen bonds (LBHBs) between His 57 and Asp 102 postulated on the
basis of spectroscopy and deuterium isotope effects. The X-ray crystal structure
of the hemiacetal adduct between Ser 195 of chymotrypsin and
N-acetyl-L-leucyl-L-phenylalanal (AcLF-CHO) has also been determined at pH 7.0.
The structure is similar to the AcLF-CF3 adduct, except for the presence of two
epimeric adducts in the R- and S-configurations at the hemiacetal carbons. In
the (R)-hemiacetal, oxygen is hydrogen bonded to His 57, not the oxyanion site.
On the basis of the downfield 1H NMR spectrum in solution, His 57 is not
protonated at Nepsilon2, and there is no LBHB at pH >7.0. Because addition of
AcLF-CHO to chymotrypsin neither releases nor takes up a proton from solution,
it is concluded that the hemiacetal oxygen of the chymotrypsin-AcLF-CHO complex
is a hydroxyl group and not attracted to the oxyanion site. The protonation
states of the hemiacetal and His 57 are explained by the high basicity of the
hemiacetal oxygen (pK(a) > 13.5) relative to that of His 57. The 13C NMR
signal for the adduct of AcLF-13CHO with chymotrypsin is consistent with a
neutral hemiacetal between pH 7 and 13. At pH <7.0, His 57 in the
AcLF-CHO-hemiacetal complex of chymotrypsin undergoes protonation at Nepsilon2
of His 57, leading to a transition of the 15.1 ppm downfield signal to 17.8 ppm.
The pK(a)s in the active sites of the AcLF-CF3 and AcLF-CHO adducts suggest an
energy barrier of 6-7 kcal x mol(-1) against ionizations that change the
electrostatic charge at the active site. However, ionizations of neutral His 57
in the AcLF-CHO-chymotrypsin adduct, or in free chymotrypsin, proceed with no
apparent barrier. Protonation of His 57 is accompanied by LBHB formation,
suggesting that stabilization by the LBHB overcomes the barrier to ionization.
On the basis of the hydration constant for AcLF-13CHO and its inhibition
constant, its K(d) is 16 microM, 8000-fold larger than the comparable value for
AcLF-CF3.
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
 |
 |
|
Literature references that cite this PDB file's key reference
|
|
|
| |
PubMed id
|
|
Reference
|
|
|
|
|
|
E.Zakharova,
M.P.Horvath,
and
D.P.Goldenberg
(2009).
Structure of a serine protease poised to resynthesize a peptide bond.
|
| |
Proc Natl Acad Sci U S A,
106,
11034-11039.
|
|
|
PDB codes:
|
|
|
|
|
|
|
|
P.Singh,
A.M.LeBeau,
H.Lilja,
S.R.Denmeade,
and
J.T.Isaacs
(2009).
Molecular insights into substrate specificity of prostate specific antigen through structural modeling.
|
| |
Proteins,
77,
984-993.
|
|
|
|
|
|
|
D.B.Berkowitz,
K.R.Karukurichi,
R.de la Salud-Bea,
D.L.Nelson,
and
C.D.McCune
(2008).
Use of Fluorinated Functionality in Enzyme Inhibitor Development: Mechanistic and Analytical Advantages.
|
| |
J Fluor Chem,
129,
731-742.
|
|
|
|
|
|
|
G.Bruylants,
C.Redfield,
and
K.Bartik
(2007).
Developments in the characterisation of the catalytic triad of alpha-chymotrypsin: Effect of the protonation state of Asp102 on the 1H NMR signals of His57.
|
| |
Chembiochem,
8,
51-54.
|
|
|
|
|
|
|
M.Brylinski,
M.Kochanczyk,
E.Broniatowska,
and
I.Roterman
(2007).
Localization of ligand binding site in proteins identified in silico.
|
| |
J Mol Model,
13,
665-675.
|
|
|
|
|
|
|
B.Liu,
C.J.Schofield,
and
R.C.Wilmouth
(2006).
Structural analyses on intermediates in serine protease catalysis.
|
| |
J Biol Chem,
281,
24024-24035.
|
|
|
PDB codes:
|
|
|
|
|
|
|
|
X.Ding,
B.F.Rasmussen,
G.A.Petsko,
and
D.Ringe
(2006).
Direct crystallographic observation of an acyl-enzyme intermediate in the elastase-catalyzed hydrolysis of a peptidyl ester substrate: Exploiting the "glass transition" in protein dynamics.
|
| |
Bioorg Chem,
34,
410-423.
|
|
|
|
|
|
|
T.K.Manojkumar,
C.Cui,
and
K.S.Kim
(2005).
Theoretical insights into the mechanism of acetylcholinesterase-catalyzed acylation of acetylcholine.
|
| |
J Comput Chem,
26,
606-611.
|
|
|
|
|
|
|
M.Shokhen,
and
A.Albeck
(2004).
Is there a weak H-bond --> LBHB transition on tetrahedral complex formation in serine proteases?
|
| |
Proteins,
54,
468-477.
|
|
|
|
|
|
|
R.M.Day,
C.J.Thalhauser,
J.L.Sudmeier,
M.P.Vincent,
E.V.Torchilin,
D.G.Sanford,
C.W.Bachovchin,
and
W.W.Bachovchin
(2003).
Tautomerism, acid-base equilibria, and H-bonding of the six histidines in subtilisin BPN' by NMR.
|
| |
Protein Sci,
12,
794-810.
|
|
|
|
|
|
|
G.G.Hammes
(2002).
Multiple conformational changes in enzyme catalysis.
|
| |
Biochemistry,
41,
8221-8228.
|
|
|
|
|
|
|
J.Overgaard,
B.Schiøtt,
F.K.Larsen,
and
B.B.Iversen
(2001).
The charge density distribution in a model compound of the catalytic triad in serine proteases.
|
| |
Chemistry,
7,
3756-3767.
|
|
|
|
|
|
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
