Allosteric changes of thrombin catalytic site induced by interaction of bothrojaracin with anion-binding exosites I and II

Bothrojaracin, a 27-kDa C-type lectin from Bothrops jararaca venom, is a se lective and potent thrombin inhibitor (K-d = 0.6 nM) which interacts with t he two thrombin anion-binding exosites (I and II) but not with its catalyti c site. In the present study, we analyzed the allosteric effects produced i n the catalytic site by bothrojaracin binding to thrombin exosites. Opposit e effects were observed with cu-thrombin, which possesses both exosites I a nd II, and with gamma-thrombin, which lacks exosite I. On the one hand, bot hrojaracin altered both kinetic parameters K-m and k(cat) of alpha-thrombin for small synthetic substrates, resulting in an increased efficiency of al pha-thrombin catalytic activity, This effect was similar to that produced b y hirugen, a peptide based on the C-terminal hirudin sequence (residues 54- 65) which interacts exclusively with exosite I. On the other hand, bothroja racin decreased the amidolytic activity of gamma-thrombin toward chromogeni c substrates, although this effect was observed with higher concentrations of bothrojaracin than those used with alpha-thrombin. In agreement with the se observations, bothrojaracin produced opposite effects on the fluorescenc e intensity of alpha- and gamma-thrombin derivatives labeled at the active site with fluorescein-Phe-Pro-Arg-chloromethylketone. These observations su pport the conclusion that bothrojaracin binding to thrombin produces two di fferent structural changes in its active site, depending on whether it inte racts exclusively with exosite II, as seen with gamma-thrombin, or with exo site I (or both I and II) as observed with alpha-thrombin. The ability of b othrojaracin to evoke distinct modifications in the thrombin catalytic site environment when interacting with exosites I and II make this molecule an interesting tool for the study of allosteric changes in the thrombin molecu le. (C) 1999 Academic Press.