title: Peptide substrate specificity and properties of the zinc-endopeptidase activity of botulinum type B neurotoxin.
abstract: 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.
authors.affiliation: Protein Toxins Section, Centre for Applied Microbiology and Research, Salisbury, England.
authors.forename: C C
authors.initials: CC
authors.lastname: Shone
authors.forename: A K
authors.initials: AK
authors.lastname: Roberts
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001905:Botulinum Toxins / Q000378:metabolism*; D002413:Cations, Divalent; D003014:Clostridium botulinum / Q000378:metabolism*; D003300:Copper / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009498:Neurotoxins / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D050683:R-SNARE Proteins; D051381:Rats; D013379:Substrate Specificity; D015032:Zinc / Q000494:pharmacology
chemical_list: D002413:Cations, Divalent; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009498:Neurotoxins; D010446:Peptide Fragments; D050683:R-SNARE Proteins; D003300:Copper; D008666:Metalloendopeptidases; D001905:Botulinum Toxins; D015032:Zinc