Botulinum neurotoxins (BoNTs A-G) are zinc metalloendoproteases that exhibit extraordinary specificities for proteins involved in neurotransmitter release. In view of the extreme toxicities of these molecules, their applications in human medicine, and potential for misuse, it is of considerable importance to elucidate the mechanisms underlying substrate recognition and to develop inhibitors, with the ultimate goal of obtaining anti-botulinum drugs. We synthesized peptides based on vesicle-associated membrane protein (VAMP) to investigate the substrate requirements of BoNT F, which cleaves VAMP between residues Q58 and K59. The minimum substrate was a peptide containing VAMP residues 32-65, which includes only one of the two VAMP structural motifs thought to be required for botulinum substrate recognition. BoNT F exhibited a strict requirement for residues D57 (P 2 ), K59 (P 1 ′), and L60 (P 2 ′), but peptides containing substitutions for R56 (P 3 ), Q58 (P 1 ), and S61 (P 3 ′) were cleaved. Therefore, the P 2 , P 1 ′, and P 2 ′ residues of VAMP are of paramount importance for BoNT F substrate recognition near the scissile bond. K i values of uncleavable analogues were similar to K m values of the substrate, suggesting that substrate discrimination occurs at the cleavage step, not at the initial binding step. We then synthesized inhibitors of BoNT F that incorporated D-cysteine in place of glutamine 58, exhibited K i values of 1-2 nM, and required binding groups on the N-terminal but not the C-terminal side of the zinc ligand. The latter characteristic distinguishes BoNT F from other zinc metalloendoproteases, including BoNTs A and B.The clostridial neurotoxins consist of tetanus toxin, secreted by Clostridium tetani, and the seven serologically distinct botulinum toxins, designated types A-G, produced by various strains of Clostridium botulinum, Clostridium baratii, and Clostridium butyricum (1-3). They are the most toxic substances known (4). Each neurotoxin is synthesized as a single-chain protein with an M r of ∼150000. Endogenous proteases then act to produce the dichain structure, consisting of an M r ∼ 100 000 heavy chain and an M r ∼ 50 000 light chain, covalently linked by a disulfide bond. The dichain is thought to be the active form of the toxin (1). In the United States, there are ∼200 cases of human botulism each year, due to ingestion of toxin-contaminated food, wound infections, or colonization of the intestinal tract by C. botulinum (5-7). However, much of the current interest in BoNTs 1 stems from their use as tools in research on the mechanisms of neurotransmission (1,8,9), an ever-expanding number of applications in the treatment of human muscle dysfunctions (10-13), and their potential for use as bioterrorist weapons (14).Intoxication resulting in the flaccid paralysis of botulism or the spastic paralysis of tetanus has been described as a four-step process, consisting of (1) toxin binding to specific receptors on neurons, (2) receptor-mediated toxin endocytosis, (3) translocation th...