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PDBsum entry 1awh
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Complex (protease/inhibitor)
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PDB id
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1awh
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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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Novel natural product 5,5-Trans-Lactone inhibitors of human alpha-Thrombin: mechanism of action and structural studies.
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Authors
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M.P.Weir,
S.S.Bethell,
A.Cleasby,
C.J.Campbell,
R.J.Dennis,
C.J.Dix,
H.Finch,
H.Jhoti,
C.J.Mooney,
S.Patel,
C.M.Tang,
M.Ward,
A.J.Wonacott,
C.W.Wharton.
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Ref.
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Biochemistry, 1998,
37,
6645-6657.
[DOI no: ]
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PubMed id
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Abstract
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High-throughput screening of methanolic extracts from the leaves of the plant
Lantana camara identified potent inhibitors of human alpha-thrombin, which were
shown to be 5,5-trans-fused cyclic lactone euphane triterpenes [O'Neill et al.
(1998) J. Nat. Prod. (submitted for publication)]. Proflavin displacement
studies showed the inhibitors to bind at the active site of alpha-thrombin and
alpha-chymotrypsin. Kinetic analysis of alpha-thrombin showed tight-binding
reversible competitive inhibition by both compounds, named GR133487 and
GR133686, with respective kon values at pH 8.4 of 1.7 x 10(6) s-1 M-1 and 4.6 x
10(6) s-1 M-1. Electrospray ionization mass spectrometry of thrombin/inhibitor
complexes showed the tight-bound species to be covalently attached, suggesting
acyl-enzyme formation by reaction of the active-site Ser195 with the
trans-lactone carbonyl. X-ray crystal structures of alpha-thrombin/GR133686 (3.0
A resolution) and alpha-thrombin/GR133487 (2.2 A resolution) complexes showed
continuous electron density between Ser195 and the ring-opened lactone carbonyl,
demonstrating acyl-enzyme formation. Turnover of inhibitor by alpha-thrombin was
negligible and mass spectrometry of isolated complexes showed that reversal of
inhibition occurs by reformation of the trans-lactone from the acyl-enzyme. The
catalytic triad appears undisrupted and the inhibitor carbonyl occupies the
oxyanion hole, suggesting the observed lack of turnover is due to exclusion of
water for deacylation. The acyl-enzyme inhibitor hydroxyl is properly positioned
for nucleophilic attack on the ester carbonyl and therefore relactonization;
furthermore, the higher resolution structure of alpha-thrombin/GR133487 shows
this hydroxyl to be effectively superimposable with the recently proposed
deacylating water for peptide substrate hydrolysis [Wilmouth, R. C., et al.
(1997) Nat. Struct.Biol. 4, 456-462], suggesting the alpha-thrombin/GR133487
complex may be a good model for this reaction.
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