Abstract
Type A botulinum neurotoxin, a zinc-dependent endoproteinase that selectively cleaves the neuronal protein SNAP-25, can also cleave relatively short peptides. We found that bovine and other serum albumins stimulated the type A-catalyzed hydrolysis of synthetic peptide substrates, through a direct effect on the kinetic constants of the reaction. Furthermore, with bovine serum albumin in the assays, the optimum substrate size was 16 residues (11 on the amino-terminal side of the cleavage site and 5 on the carboxy-terminal side). To further investigate the catalytic requirements of the neurotoxin, peptides were synthesized with various amino acid substitutions at the P5 through P5′ substrate sites. Changes at all of these locations affected values for both k cat and K m. Substitutions at the P2, P1′, and P2′ sites had more pronounced effects on hydrolysis rates than did substitutions at the P1 site. Enzyme–substrate interactions at the P3′ threonine probably involved the side-chain methyl group rather than the hydroxyl group. Replacing the P2′ alanine with leucine eliminated detectable hydrolysis, but not binding, since this peptide was an inhibitor. A negatively charged residue was preferred at P5, but not at P4. The data indicate that type A botulinum neurotoxin has an extended substrate recognition region and a requirement for arginine as the P1′ residue.
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Schmidt, J.J., Bostian, K.A. Endoproteinase Activity of Type A Botulinum Neurotoxin: Substrate Requirements and Activation by Serum Albumin. J Protein Chem 16, 19–26 (1997). https://doi.org/10.1023/A:1026386710428
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DOI: https://doi.org/10.1023/A:1026386710428