Shone C C, Roberts A K
Protein Toxins Section, Centre for Applied Microbiology and Research, Salisbury, England.
Eur J Biochem. 1994 Oct 1;225(1):263-70. doi: 10.1111/j.1432-1033.1994.00263.x.
Clostridium botulinum type B neurotoxin has been shown to be a zinc endopeptidase specific for vesicle-associated membrane protein (VAMP). A synthetic peptide of human/rat VAMP-2 [VAMP-2-(60-94)] is cleaved by the neurotoxin with the same specificity as that demonstrated for the membrane-associated protein (at the Gln76-Phe77 bond) and has been used to study the properties of the endopeptidase activity of the neurotoxin. Cleavage of the VAMP-2 peptide was demonstrated by both botulinum type B neurotoxin (Km = 3.3 x 10(-4) M) and by its purified light subunit (Km = 3.5 x 10(-4) M). The endopeptidase displayed a pH optimum of 7.0-7.5 and was inhibited by greater than 0.2 M NaCl and greater than 0.05 M sodium phosphate. Neurotoxin which had been inactivated by dialysis against EDTA could be re-activated by incubation with various divalent cations, notably Zn2+ and Cu2+. The substrate specificity of botulinum type B neurotoxin was studied using various analogues of VAMP-2 (60-94). The neurotoxin cleaved selectively to the N-terminal side of phenylalanine and tyrosine; no activity was observed with either leucine, valine or alanine in the P'1 position. The properties of the P1 amino acid were less critical; the neurotoxin cleaving the C-terminus of glutamine, asparagine and alanine. A substrate analogue with valine in the P1 position corresponding to the sequence of rat VAMP-1 was not cleaved. The rate of cleavage of a substrate analogue representing the sequence of human VAMP-1, however, was more than twofold that of the VAMP-2 peptide. The properties and substrate specificity of botulinum type B neurotoxin suggest that the toxin represents a novel class of endopeptidase which requires a specific peptide substrate conformation for the expression of proteolytic activity.
B型肉毒杆菌神经毒素已被证明是一种对囊泡相关膜蛋白（VAMP）具有特异性的锌内肽酶。人/大鼠VAMP-2的合成肽[VAMP-2-(60-94)]被神经毒素切割，其特异性与膜相关蛋白相同（在Gln76-Phe77键处），并已用于研究神经毒素内肽酶活性的特性。B型肉毒杆菌神经毒素（Km = 3.3×10(-4) M）及其纯化的轻链（Km = 3.5×10(-4) M）均证明能切割VAMP-2肽。该内肽酶的最适pH为7.0-7.5，大于0.2 M的NaCl和大于0.05 M的磷酸钠可抑制其活性。经EDTA透析失活的神经毒素可通过与各种二价阳离子（尤其是Zn2+和Cu2+）孵育而重新激活。使用VAMP-2 (60-94)的各种类似物研究了B型肉毒杆菌神经毒素的底物特异性。神经毒素选择性地在苯丙氨酸和酪氨酸的N端一侧切割；在P'1位置的亮氨酸、缬氨酸或丙氨酸均未观察到活性。P1氨基酸的性质不太关键；神经毒素切割谷氨酰胺、天冬酰胺和丙氨酸的C端。对应于大鼠VAMP-1序列且在P1位置含有缬氨酸的底物类似物未被切割。然而，代表人类VAMP-1序列的底物类似物的切割速率是VAMP-2肽的两倍多。B型肉毒杆菌神经毒素的特性和底物特异性表明，该毒素代表了一类新型的内肽酶，其蛋白水解活性的表达需要特定的肽底物构象。
