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= 3.3×10−4M) and by its purified light subunit (Km= 3.5×10−4M). 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 reactivated 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. Copyright © 1994, Wiley Blackwell. All rights reserved