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PDBsum entry 2h1u
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References listed in PDB file
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Key reference
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Title
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Structural analyses on intermediates in serine protease catalysis.
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Authors
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B.Liu,
C.J.Schofield,
R.C.Wilmouth.
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Ref.
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J Biol Chem, 2006,
281,
24024-24035.
[DOI no: ]
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PubMed id
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Abstract
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Although the subject of many studies, detailed structural information on aspects
of the catalytic cycle of serine proteases is lacking. Crystallographic analyses
were performed in which an acyl-enzyme complex, formed from elastase and a
peptide, was reacted with a series of nucleophilic dipeptides. Multiple analyses
led to electron density maps consistent with the formation of a tetrahedral
species. In certain cases, apparent peptide bond formation at the active site
was observed, and the electron density maps suggested production of a cis-amide
rather than a trans-amide. Evidence for a cis-amide configuration was also
observed in the noncovalent complex between elastase and an
alpha1-antitrypsin-derived tetrapeptide. Although there are caveats on the
relevance of the crystallographic data to solution catalysis, the results enable
detailed proposals for the pathway of the acylation step to be made. At least in
some cases, it is proposed that the alcohol of Ser-195 may preferentially attack
the carbonyl of the cis-amide form of the substrate, in a stereoelectronically
favored manner, to give a tetrahedral oxyanion intermediate, which undergoes
N-inversion and/or C-N bond rotation to enable protonation of the leaving group
nitrogen. The mechanistic proposals may have consequences for protease
inhibition, in particular for the design of high energy intermediate analogues.
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Figure 3.
FIGURE 3. Stereoviews of the active site of PPE complexed
with BCM7 and different dipeptides showing the results of the pH
jump (pH 9, 30s) of the Lys-Ser structure shown in Fig. 1b (a)
and the results of the pH jumps of the Arg-Phe structure shown
in Fig. 2d (one pH jump (pH 9, 30 s) (b) and a second pH jump
(pH 9, 28 s) (c)). The color scheme and contouring levels for
the atoms and maps are as in Fig. 1a. The resolution of the
structures is 1.8, 1.7, and 1.9 Å, respectively.
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Figure 5.
FIGURE 5. Two proposed forms of the first tetrahedral
intermediate (A and B) interchangeable via N-inversion and/or
rotation about the C-N bond. In A, the P1' nitrogen lone pair
projects away from His-57, and in B it projects toward it. In
intermediate B, R and R' refer to either hydrogen or the P1'
residue, depending on whether N-inversion has occurred. The
amide nitrogen of Ser-195, which forms part of the oxyanion
hole, is not shown for clarity; from the viewpoint shown, it
lies behind the plane of the picture. Note that steric
constraints mean that the conformation in which the nitrogen
lone pair is exactly coplanar with the C-O(Ser-195) bond is
unlikely.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2006,
281,
24024-24035)
copyright 2006.
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Secondary reference #1
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Title
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Mass spectrometry reveals elastase inhibitors from the reactive centre loop of alpha1-Antitrypsin.
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Authors
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P.A.Wright,
A.A.Rostom,
C.V.Robinson,
C.J.Schofield.
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Ref.
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Bioorg Med Chem Lett, 2000,
10,
1219-1221.
[DOI no: ]
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PubMed id
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