TYPE-A BOTULINUM NEUROTOXIN PROTEOLYTIC ACTIVITY - DEVELOPMENT OF COMPETITIVE INHIBITORS AND IMPLICATIONS FOR SUBSTRATE-SPECIFICITY AT THE S-1' BINDING SUBSITE

Type A botulinum neurotoxin (botox A) is a zinc metalloprotease that c leaves only one peptide bond in the synaptosomal protein, SNAP-25. Sin gle-residue changes in a 17-residue substrate peptide mere used to dev elop the first specific, competitive inhibitors of its proteolytic act ivity. 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 cystei ne 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 s ubstrate, 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 disp layed an unusual requirement for arginine as the P-1' inhibitor residu e, demonstrating that the S-1' binding subsite of botox A is dissimila r 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.