1994
DOI: 10.1111/j.1432-1033.1994.00263.x
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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 h u m d r a t 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 (… Show more

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Cited by 101 publications
(121 citation statements)
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“…In the BoNT F minimum substrate, 27 of the required residues are on the N-terminal side of the scissile bond, while only seven are needed on the C-terminal side. The latter situation is similar to that of the BoNT A substrate, with only five residues C-terminal to the cleavage site (31), but differs from that of BoNT B, where 18 residues are needed (29).…”
Section: Discussionmentioning
confidence: 78%
“…In the BoNT F minimum substrate, 27 of the required residues are on the N-terminal side of the scissile bond, while only seven are needed on the C-terminal side. The latter situation is similar to that of the BoNT A substrate, with only five residues C-terminal to the cleavage site (31), but differs from that of BoNT B, where 18 residues are needed (29).…”
Section: Discussionmentioning
confidence: 78%
“…Even larger substrates (>50 residues) are required by tetanus toxin and botulinum type A neurotoxin. Surprisingly, in a study of the peptide substrate specificity of BoNT/B only one VAMP residue at the cleavage site (Phe77) was found to be critical to the endopeptidase activity of the toxin [14]. These data suggest that determinants other than the cleavage site sequence are required to account for the neurotoxin's highly specific proteolytic activity.…”
Section: Introductionmentioning
confidence: 95%
“…The strongest cleavage defect in vitro resulted from the S75F mutation at the P2 position; Ϸ1,500-fold excess B-LC was required to achieve 50% cleavage. Prior studies have shown that S75A and S75T substitutions, which are more conservative than our S75F substitution, exert modest defects on the rate of Sb fragment cleavage in vitro (9). The strong cleavage defect conferred by S75F leads us to suggest that B-LC does not recognize Sb when it contains residues with large and͞or aromatic side chains at the P2 position.…”
Section: Discussionmentioning
confidence: 85%
“…Additional Sb residues are probably required for efficient B-LC recognition. Indeed, we were surprised when amino acid substitutions did not appear at the PЈ1 position (F73) of Snc2͞Sb͞Snc2 by yeast selection; Sb PЈ1 residue (F77) was implicated previously in B-LC recognition (9). Absence of PЈ1 substitutions in our yeast genetic selection can be explained by the fact that when we constructed an F73A substitution in Snc2͞Sb͞Snc2 and expressed it in yeast, the mutant SNARE did not support cell growth (data not shown).…”
Section: Discussionmentioning
confidence: 98%
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