Cornille F, Martin L, Lenoir C, Cussac D, Roques B P, Fournie-Zaluski M C
Département de Pharmacochimie Moléculaire et Structurale, U266 INSERM, URA D1500 CNRS, Université René Descartes, UFR des Sciences Pharmaceutiques et Biologiques, 75270 Paris Cedex 06, France.
J Biol Chem. 1997 Feb 7;272(6):3459-64. doi: 10.1074/jbc.272.6.3459.
The light chain (L chain) of tetanus neurotoxin (TeNT) has been shown to have been endowed with zinc endopeptidase activity, selectively directed toward the Gln76-Phe77 bond of synaptobrevin, a vesicle-associated membrane protein (VAMP) critically involved in neuroexocytosis. In previous reports, truncations at the NH2 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 NH2- and COOH-terminal peptides of synaptobrevin, S 27-55 (S1) and S 82-93 (S2), 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 S1 and S2 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.
破伤风神经毒素（TeNT）的轻链（L链）已被证明具有锌内肽酶活性，可选择性地作用于突触小泡蛋白（一种与囊泡相关的膜蛋白，即VAMP，在神经递质释放过程中起关键作用）的Gln76 - Phe77键。在之前的报道中，突触小泡蛋白氨基端和羧基端的截短突变表明，突触小泡蛋白的39 - 88序列是TeNT的最小底物，这表明要么需要突触小泡蛋白具有明确的三维结构，要么远离切割位点的残基在底物水解机制中起作用。在本研究中，向突触小泡蛋白片段S 56 - 81添加突触小泡蛋白的氨基端和羧基端肽段S 27 - 55（S1）和S 82 - 93（S2），使得TeNT能够切割后一种肽段。表面等离子体共振实验和动力学常数测定表明，这似乎是由S1和S2与TeNT上存在的互补位点同时结合介导的激活过程导致的。所有这些结果都支持TeNT对突触小泡蛋白进行别构位点控制的水解作用，这可能涉及毒素的构象变化。这可以解释TeNT以及可能还有肉毒杆菌神经毒素的高度底物特异性。
