Cornille F, Goudreau N, Ficheux D, Niemann H, Roques B P
Département de Pharmacochimie Moléculaire et Structurale, CNRS URA D1500-INSERM U266, Université Paris V, France.
Eur J Biochem. 1994 May 15;222(1):173-81. doi: 10.1111/j.1432-1033.1994.tb18855.x.
A 93-residue peptide corresponding to the cytosolic domain of a human vesicle associated membrane protein (VAMP or synaptobrevin) has been prepared by solid-phase peptide synthesis in order to investigate the proteolytic activity of the tetanus toxin light chain (TeTx L chain). This protein has been recently reported to inactivate the neuronal rat synaptobrevin II by proteolysis. We show in this study that the synthetic human synaptobrevin II 1-93 (Syb II 1-93) as well as an N-terminus-shortened 69-residue peptide (Syb II 25-93) were cleaved selectively at the Gln76-Phe77 peptide bond by TeTx L chain while shorter peptides were not. A Michaelis constant Km = 192 +/- 2 microM and a catalytic constant kcat = 0.5 min-1 were found for the 93-residue peptide. A neutral optimum pH for the cleavage rate, an inhibition by preincubation of the toxin with well known nonspecific inhibitors of metallopeptidases as well as a zinc-dependent enzyme activity suggest that TeTx belongs to the zinc endopeptidase family. Moreover an activation by reducing agents and an inhibition by cysteine-modifying chemical reagents indicate a critical thiol dependency. Among several specific inhibitors of zinc endopeptidases tested, none could inhibit TeTx L chain even at high concentration. Structural studies by 600-MHz 1H-NMR showed that in water or dimethylsulfoxide the peptide Syb II 1-93 and shorter fragments did not present well defined conformations. Nevertheless protein-protein interactions have been shown for the peptides Syb II 1-93 and 25-93 but not for Syb II 51-93, a fragment not cleaved by TeTx L chain.
为了研究破伤风毒素轻链（TeTx L链）的蛋白水解活性，通过固相肽合成制备了一种对应于人囊泡相关膜蛋白（VAMP或突触小泡蛋白）胞质结构域的93个残基的肽。最近有报道称该蛋白通过蛋白水解作用使大鼠神经元突触小泡蛋白II失活。我们在本研究中表明，合成的人突触小泡蛋白II 1-93（Syb II 1-93）以及N端缩短的69个残基的肽（Syb II 25-93）被TeTx L链在Gln76-Phe77肽键处选择性切割，而较短的肽则未被切割。对于93个残基的肽，测得米氏常数Km = 192±2 microM，催化常数kcat = 0.5 min-1。切割速率的最适pH为中性，毒素与金属肽酶的已知非特异性抑制剂预孵育会产生抑制作用，以及锌依赖性酶活性表明TeTx属于锌内肽酶家族。此外，还原剂可激活该酶，而半胱氨酸修饰化学试剂可抑制该酶，这表明存在关键的巯基依赖性。在所测试的几种锌内肽酶特异性抑制剂中，即使在高浓度下也没有一种能够抑制TeTx L链。600-MHz 1H-NMR的结构研究表明，在水或二甲基亚砜中，肽Syb II 1-93和较短的片段没有呈现出明确的构象。然而，已显示肽Syb II 1-93和25-93存在蛋白质-蛋白质相互作用，而未被TeTx L链切割的片段Syb II 51-93则没有这种相互作用。
