1awf: Difference between revisions

==Overview==

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.

==About this Structure==

[[Category: trypsin like proteinase]]