Type A botulinum neurotoxin proteolytic activity:: development of competitive inhibitors and implications for substrate specificity at the S1′ binding subsite

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 mere 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 mere not cleaved and mere inhibitors. Peptides with either D- or L-cysteine as the N-terminus, followed by the last six residues of the substrate, mere the most effective inhibitors, each with a Ki value of 2 mu M. 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. (C) 1998 Federation of European Biochemical Societies.