Abstract
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.
Highlights
The ‘‘anthrax letter’’ scare in the fall of 2001 [1] generated renewed interest in finding remedies to real and perceived bio threat agents
We have demonstrated that a full length light chain (Lc) free from rest of the botulinum neurotoxin (BoNT)/A molecule is the most catalytically active species [25]
17-Mer substrate Previously, we reported that a light chain of serotype A (LcA) preparation solubilized from inclusion bodies behaved very similar to that of whole BoNT/A toxin when assayed with the synthetic 17-mer substrate [34]
Summary
The ‘‘anthrax letter’’ scare in the fall of 2001 [1] generated renewed interest in finding remedies to real and perceived bio threat agents. It is very important that activity of one standard LcA catalyst be determined using several of the currently used substrates, so that the effects of various additives on the rate of the reaction can be evaluated Results obtained from such a study will allow a direct comparison of the properties of LcA and the substrates for a more realistic evaluation of inhibitor screening. In this backdrop, the current investigation compares the substrate properties of the 17-mer, the 66-mer, and the full length SNAP25 with the most active BoNT/A catalyst under near identical assay conditions. We show that the reaction time has a profound effect on the enzyme constants, and the full length SNAP25 is by far the best substrate that yields the lowest Km and highest kcat values
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