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 were used to develop the first specific, competitive inhibitors of its proteolytic activity. Substrate analog peptides with P4, P3, P2' or P3' cysteine were readily hydrolyzed by the toxin, but those with P1 or P2 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 Ki value of 2 microM. 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 P1' inhibitor residue, demonstrating that the S1' 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.
Toxinology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702-5011, USA. dr._James_Schmidt_at_usamriid5_ftdetrck@ftdetrck-ccmail.army.mil
J J
JJ
Schmidt
R G
RG
Stafford
K A
KA
Bostian
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D019274:Botulinum Toxins, Type A / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D006868:Hydrolysis; D008565:Membrane Proteins; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity / Q000187:drug effects; D050825:Synaptosomal-Associated Protein 25
D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009842:Oligopeptides; D050825:Synaptosomal-Associated Protein 25; D001120:Arginine; D008666:Metalloendopeptidases; D019274:Botulinum Toxins, Type A