Cleavage of SNAP25 and its shorter versions by the protease domain of serotype A botulinum neurotoxin.
Various substrates, catalysts, and assay methods are currently used to screen inhibitors for their effect on the proteolytic activity of botulinum neurotoxin. As a result, significant variation exists in the reported results. Recently, we found that one source of variation was the use of various catalysts, and have therefore evaluated its three forms. In this paper, we characterize three substrates under near uniform reaction conditions using the most active catalytic form of the toxin. Bovine serum albumin at varying optimum concentrations stimulated enzymatic activity with all three substrates. Sodium chloride had a stimulating effect on the full length synaptosomal-associated protein of 25 kDa (SNAP25) and its 66-mer substrates but had an inhibitory effect on the 17-mer substrate. We found that under optimum conditions, full length SNAP25 was a better substrate than its shorter 66-mer or 17-mer forms both in terms of kcat, Km, and catalytic efficiency kcat/Km. Assay times greater than 15 min introduced large variations and significantly reduced the catalytic efficiency. In addition to characterizing the three substrates, our results identify potential sources of variations in previous published results, and underscore the importance of using well-defined reaction components and assay conditions.
Department of Cell Biology and Biochemistry, Molecular and Translational Sciences Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, United States of America.
Rahman M
RM
Mizanur
Department of Cell Biology and Biochemistry, Molecular and Translational Sciences Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, United States of America.
Robert G
RG
Stafford
Department of Cell Biology and Biochemistry, Molecular and Translational Sciences Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, United States of America.
S Ashraf
SA
Ahmed
D019274:Botulinum Toxins, Type A / Q000037:antagonists & inhibitors / Q000737:chemistry*; D002712:Chlorides / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry; D059748:Proteolysis; D012710:Serum Albumin, Bovine / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry; D013379:Substrate Specificity; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry*; D017967:Zinc Compounds / Q000737:chemistry
D002712:Chlorides; D010446:Peptide Fragments; C494208:SNAP25 protein, human; D050825:Synaptosomal-Associated Protein 25; D017967:Zinc Compounds; D012710:Serum Albumin, Bovine; D012965:Sodium Chloride; C016837:zinc chloride; D019274:Botulinum Toxins, Type A; D004229:Dithiothreitol