C‑Terminus of Botulinum A Protease Has Profound and Unanticipated Kinetic Consequences upon the Catalytic Cleft

Botulinum neurotoxins (BoNTs) are among the most deadly poisons known, though ironically, they also are of great therapeutic utility. A number of research programs have been initiated to discover small molecule inhibitors of BoNTs metalloprotease activity. Many, though not all, of these programs have screened against a truncated and more stable form of the enzyme, that possesses comparable catalytic properties to the full length enzyme. Interestingly, several classes of inhibitors, notably the hydroxamates, display a large shift in potency between the two enzyme forms. In this report we compare the kinetics of active-site, α-exosite and β-exosite inhibitors versus truncated and full length enzyme. Molecular dynamics simulations conducted with the truncated and homology models of the full length BoNT LC/A indicate the flexibility of the C-terminus of the full length enzyme is responsible for the potency shifts of active-site proximally binding inhibitors while distal binding (α-exosite) inhibitors remain equipotent.