1AWF

NOVEL COVALENT THROMBIN INHIBITOR FROM PLANT EXTRACT

1AWF の概要

• エントリーDOI: 10.2210/pdb1awf/pdb
• 分子名称: ALPHA THROMBIN, HIRUGEN, R3-ACETOXY-17-(1-FORMYL-5-METHYL-3-OXO-HEX-4-ENYL)-12,16-DIHYDROXY-14-HYDROXYMETHYL-4,10,13-TRIMETHYL-2,3,4,5,6,9,10,11,12,13,14,15,16,17-TETRADECAHYDRO-1H-CYCLOPENTA[A]PHENANTHRENE-4-CARBOXYLIC ACID IDOPYRANOSYL ESTER, ... (5 entities in total)
• 機能のキーワード: proteinase, blood coagulation, trypsin like proteinase, complex (protease-inhibitor), hydrolase-hydrolase inhibitor complex, hydrolase/hydrolase inhibitor
• 由来する生物種: Hirudo medicinalis (medicinal leech); 詳細
• 細胞内の位置: Secreted, extracellular space: P00734 P00734
• タンパク質・核酸の鎖数: 3
• 化学式量合計: 35976.99

構造登録者

Jhoti, H.,Cleasby, A.,Wonacott, A. (登録日: 1997-10-02, 公開日: 1998-10-28, 最終更新日: 2024-10-09)

主引用文献

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

実験手法

X-RAY DIFFRACTION (2.2 Å)