title: Identification of residues surrounding the active site of type A botulinum neurotoxin important for substrate recognition and catalytic activity.
abstract: Type A botulinum neurotoxin is one of the most lethal of the seven serotypes and is increasingly used as a therapeutic agent in neuromuscular dysfunctions. Its toxic function is related to zinc-endopeptidase activity of the N-terminal light chain (LC) on synaptosome-associated protein-25 kDa (SNAP-25) of the SNARE complex. To understand the determinants of substrate specificity and assist the development of strategies for effective inhibitors, we used site-directed mutagenesis to investigate the effects of 13 polar residues of the LC on substrate binding and catalysis. Selection of the residues for mutation was based on a computational analysis of the three-dimensional structure of the LC modeled with a 17-residue substrate fragment of SNAP-25. Steady-state kinetic parameters for proteolysis of the substrate fragment were determined for a set of 16 single mutants. Of the mutated residues non-conserved among the serotypes, replacement of Arg-230 and Asp-369 by polar or apolar residues resulted in drastic lowering of the catalytic rate constant (k(ca)), but had less effect on substrate affinity (K(m)). Substitution of Arg-230 with Lys decreased the catalytic efficiency (k(cat)/K(m)) by 50-fold, whereas replacement by Leu yielded an inactive protein. Removal of the electrostatic charge at Asp-369 by mutation to Asn resulted in 140-fold decrease in k(cat)/K(m). Replacement of other variable residues surrounding the catalytic cleft (Glu-54, Glu-63, Asn-66, Asp-130, Asn-161, Glu-163, Glu-170, Glu-256), had only marginal effect on decreasing the catalytic efficiency, but unexpectedly the substitution of Lys-165 with Leu resulted in fourfold increase in k(cat)/K(m). For comparison purposes, two conserved residues Arg-362 and Tyr-365 were investigated with substitutions of Leu and Phe, respectively, and their catalytic efficiency decreased 140- and 10-fold, respectively, whereas substitution of the tyrosine ring with Asn abolished activity. The altered catalytic efficiencies of the mutants were not due to any significant changes in secondary or tertiary structures, or in zinc content and thermal stability. We suggest that, despite the large minimal substrate size for catalysis, only a few non-conserved residues surrounding the active site are important to render the LC competent for catalysis or provide conformational selection of the substrate.
authors.affiliation: Department Molecular Biology, Integrated Toxicology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702, USA. syed.ahmed@amedd.army.mil
authors.forename: S Ashraf
authors.initials: SA
authors.lastname: Ahmed
authors.forename: Mark A
authors.initials: MA
authors.lastname: Olson
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Ludivico
authors.forename: Janice
authors.initials: J
authors.lastname: Gilsdorf
authors.forename: Leonard A
authors.initials: LA
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D019274:Botulinum Toxins, Type A / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002384:Catalysis; D002942:Circular Dichroism; D003014:Clostridium botulinum / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D009498:Neurotoxins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017433:Protein Structure, Secondary; D013379:Substrate Specificity
chemical_list: D009498:Neurotoxins; D019274:Botulinum Toxins, Type A