Type A botulinum neurotoxin proteolytic activity: development of competitive inhibitors and implications for substrate specificity at the S 1′ binding subsite

Abstract

Type A botulinum neurotoxin (botox A) is a zinc metalloprotease that cleaves only one peptide bond in the synaptosomal protein, SNAP-25. Single-residue changes in a 17-residue substrate peptide were used to develop the first specific, competitive inhibitors of its proteolytic activity. Substrate analog peptides with P 4, P 3, P 2′ or P 3′ cysteine were readily hydrolyzed by the toxin, but those with P 1 or P 2 cysteine were not cleaved and were inhibitors. Peptides with either d- or l-cysteine as the N-terminus, followed by the last six residues of the substrate, were the most effective inhibitors, each with a K i value of 2 μM. Elimination of the cysteine sulfhydryl group yielded much less effective inhibitors, suggesting that inhibition was primarily due to binding of the active-site zinc by the sulfhydryl group. Botox A displayed an unusual requirement for arginine as the P 1′ inhibitor residue, demonstrating that the S 1′ binding subsite of botox A is dissimilar to those of most other zinc metalloproteases. This characteristic is an important element in shaping the substrate specificity of botox A.