No abstract

The light chain (L chain) of tetanus neurotoxin (TeNT) has been shown to have been endowed with zinc endopeptidase activity, selectively directed toward the Gln 76 -Phe 77 bond of synaptobrevin, a vesicle-associated membrane protein (VAMP) critically involved in neuroexocytosis. In previous reports, truncations at the NH 2 and COOH terminus of synaptobrevin have shown that the sequence 39 -88 of synaptobrevin is the minimum substrate of TeNT, suggesting either the requirement of a well defined three-dimensional structure of synaptobrevin or a role in the mechanism of substrate hydrolysis for residues distal from the cleavage site. In this study, the addition of NH 2 -and COOH-terminal peptides of synaptobrevin, S 27-55 (S 1 ) and S 82-93 (S 2 ), to the synaptobrevin fragment S 56 -81 allowed the cleavage of this latter peptide by TeNT to occur. This appears to result from an activation process mediated by the simultaneous binding of S 1 and S 2 with complementary sites present on TeNT as shown by surface plasmon resonance experiments and the determination of kinetic constants. All these results favor an exosite-controlled hydrolysis of synaptobrevin by TeNT, probably involving a conformational change of the toxin. This could account for the high degree of substrate specificity of TeNT and, probably, botulinum neurotoxins.