1AWH

NOVEL COVALENT THROMBIN INHIBITOR FROM PLANT EXTRACT

Summary for 1AWH

• Entry DOI: 10.2210/pdb1awh/pdb
• Descriptor: ALPHA THROMBIN, 3-ACETOXY-17-(1-FORMYL-5-METHYL-3-OXO-HEX-4-ENYL)-16-HYDROXY-4,10,13,14-TETRAMETHYL-2,3,4,5,6,9,10,11,12,13,14,15,16,17-TETRADECAHYDRO-1H-CYCLOPENTA[A]PHENANTHRENE-4-CARBOXYLIC ACID, ... (4 entities in total)
• Functional Keywords: proteinase, blood coagulation, trypsin like proteinase, complex (protease-inhibitor), complex (protease-inhibitor) complex, complex (protease/inhibitor)
• Biological source: Homo sapiens (human); More
• Cellular location: Secreted, extracellular space: P00734 P00734
• Total number of polymer chains: 4
• Total formula weight: 68838.91

Authors

Jhoti, H.,Cleasby, A.,Wonacott, A. (deposition date: 1997-10-02, release date: 1998-10-28, Last modification date: 2024-10-23)

Primary citation

Weir, M.P.,Bethell, S.S.,Cleasby, A.,Campbell, C.J.,Dennis, R.J.,Dix, C.J.,Finch, H.,Jhoti, H.,Mooney, C.J.,Patel, S.,Tang, C.M.,Ward, M.,Wonacott, A.J.,Wharton, C.W.
Novel natural product 5,5-trans-lactone inhibitors of human alpha-thrombin: mechanism of action and structural studies.
Biochemistry, 37:6645-6657, 1998

PubMed Abstract: 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.

PubMed: 9578548
DOI: 10.1021/bi972499o

Experimental method

X-RAY DIFFRACTION (3 Å)