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PDBsum entry 1uma
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Hydrolase/hydrolase inhibitor
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PDB id
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1uma
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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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Human alpha-Thrombin inhibition by the active site titrant n alpha-(N,N-Dimethylcarbamoyl)-Alpha-Azalysine p-Nitrophenyl ester: a comparative kinetic and X-Ray crystallographic study.
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Authors
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M.Nardini,
A.Pesce,
M.Rizzi,
E.Casale,
R.Ferraccioli,
G.Balliano,
P.Milla,
P.Ascenzi,
M.Bolognesi.
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Ref.
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J Mol Biol, 1996,
258,
851-859.
[DOI no: ]
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PubMed id
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Note In the PDB file this reference is
annotated as "TO BE PUBLISHED".
The citation details given above were identified by an automated
search of PubMed on title and author
names, giving a
percentage match of
93%.
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Abstract
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Kinetics for the hydrolysis of the chromogenic active site titrant N
alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester
(Dmc-azaLys-ONp) catalyzed by bovine beta-trypsin, bovine alpha-thrombin, human
alpha-thrombin, human Lys77-plasmin, human urinary kallikrein, the M(r) 33,000
and M(r) 54,000 species of human urokinase, as well as by porcine pancreatic
beta-kallikrein-A and B have been obtained between pH 6.0 and 8.0, at 21.0
degrees C. Moreover, the three dimensional structure of the human
alpha-thrombin-(hirugen).Dmc-azaLys acyl.enzyme complex has been analyzed and
refined by X-ray crystallography at 2.0 A resolution (R-factor = 0.168). As
observed for bovine beta-trypsin, the acylating inhibitor molecule is covalently
bound to the Ser195 catalytic residue, filling the human alpha-thrombin S1
primary specificity subsite with its lysyl side-group. However, the carbonyl
group of the scissile human alpha-thrombin.Dmc-azaLys acyl bond does not occupy
properly the oxyanion binding hole. At variance from the bovine
beta-trypsin.Dmc-azaLys acyl.enzyme structure, a second, not covalently bound,
inhibitor molecule, partly shielded by the 60-insertion loop of human
alpha-thrombin, is contacting the enzyme "aryl-binding site".
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Figure 1.
Figure 1. Chemical structure of
Dmc-azaLys-ONp. The O-8 and
O-12 atoms of the active site titrant,
relevant for the discussion, have
been labeled.
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Figure 2.
Figure 2. A, Time-course of p-nitrophenol release during the human a-thrombin (trace a) and bovine a-thrombin (trace
b) catalyzed hydrolysis of Dmc-azaLys-ONp at pH 6.2 and 21.0°C, with [S0]e5[E0 ]. Kinetics of p-nitrophenol release
were obtained at [Dmc-azaLys-ONp] = 5.0 × 10
2
mM; [E0] was 5.0 mM. The total amplitude of the time-course of
p-nitrophenol release (p; see equation (2)) corresponds to an absorbance change of 2.6 × 10
-2
(i.e. to an active enzyme
concentration of 5.0 mM). Values of the apparent first-order rate constant of the time-course of p-nitrophenol release (k;
s
-1
) are 5.45 s
-1
(trace a) and 3.65 s
-1
(trace b). B, Effect of the Dmc-azaLys-ONp concentration on the apparent first-order
rate constant (k) for the human a-thrombin (open circles) and bovine a-thrombin (filled circles) catalyzed hydrolysis
of the active site titrant at pH 6.2 and 21.0°C, with [S0 ]e5 [E0 ]. The continuous lines, representing the best theoretical
curves fitting the data, were calculated according to equation (3) with values of KS and k+2 given in Table 1. An average
S.E. of 27% was evaluated as the standard deviation for values of KS and k+2 . The arrows indicate the asymptotic values
of k that correspond to k+2 when [S0 ]>KS (see equation (3)). For further details, see the text.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(1996,
258,
851-859)
copyright 1996.
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Secondary reference #1
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Title
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Inhibition of serine proteinases belonging to the chymotrypsin superfamily by the cyclic thiolic compound ys3025: a comparative crystallographic study.
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Authors
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K.Djinovic carugo,
M.Rizzi,
M.Fasano,
M.Luisetti,
C.La rosa,
P.Ascenzi,
M.Bolognesi.
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Ref.
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Biochem Biophys Res Commun, 1993,
193,
32-39.
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PubMed id
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Secondary reference #2
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Title
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Refined structure of the hirudin-Thrombin complex.
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Authors
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T.J.Rydel,
A.Tulinsky,
W.Bode,
R.Huber.
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Ref.
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J Mol Biol, 1991,
221,
583-601.
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PubMed id
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