PEPTIDE SUBSTRATE-SPECIFICITY AND PROPERTIES OF THE ZINC-ENDOPEPTIDASE ACTIVITY OF BOTULINUM TYPE-B NEUROTOXIN

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 mem brane-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 (K-m = 3.3 X 10(-4)M) and by its purified light subunit (K-m 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 P-1 amino acid were less critical; the neurotoxin cleaving the C-terminus of glutamine, asparagine and alanine. A substrate analogue with valine in the P-1 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.