[Show abstract][Hide abstract] ABSTRACT: The light chain of botulinum neurotoxin A (BoNT/A-LC) is a Zn-dependent protease that specifically cleaves SNAP25 of the SNARE complex, thereby impairing vesicle fusion and neurotransmitter release at neuromuscular junctions. The C-terminus of SNAP25 (residues 141-206) retains full activity for BoNT/A-LC-catalyzed cleavage at P1-P1' (Gln197-Arg198). Using the structure of a complex between the C-terminus of SNAP25 and BoNT/A-LC as a model to design SNAP25-derived pseudosubstrate inhibitors (SNAPIs) that prevent presentation of the scissile bond to the active site, we introduced multiple His residues to replace Ala-Asn-Gln-Arg (residues 195-198) at the substrate cleavage site, with the intent to identify possible side-chain interactions with the active site Zn. We also introduced multiple Gly residues between the P1-P1' residues to explore the spatial tolerance within the active-site cleft. Using a FRET substrate YsCsY, we compared a series of SNAPIs for inhibition of BoNT/A-LC. Among the SNAPIs tested, several known cleavage-resistant, single-point mutants of SNAP25 were poor inhibitors, with most of the mutants losing binding affinity. Replacement with His at the active site did not improve inhibition over wildtype substrate. In contrast, Gly-insertion mutants were not only resistant to cleavage, but also surprisingly showed enhanced affinity for BoNT/A-LC. Two of the Gly-insertion mutants exhibited 10-fold lower IC₅₀ values than the wildtype 66-mer SNAP25 peptide. Our findings illustrate a scenario, where the induced fit between enzyme and bound pseudosubstrate fails to produce the strain and distortion required for catalysis to proceed.