Type A botulinum neurotoxin proteolytic activity: development of competitive inhibitors and implications for substrate specificity at the S1′ binding subsite

FEBS Letters

Stafford, Robert G.¹  Bostian, Karen A.¹  Schmidt, James J.¹ 

[1] Toxinology Division, United States Army Medical Research Institute of Infectious Diseases, 1425 Porter St., Fort Detrick, MD 21702-5011, USA

关键词: Botulinum neurotoxin;    Inhibitor;    Protease;    Botox;    Clostridium botulinum neurotoxin (the subsequent capital letter indicates the serotype);   

DOI  :  10.1016/S0014-5793(98)01041-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 K _i value of 2 μM. 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.

【 授权许可】

Unknown