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Molecular basis of the endopeptidase activity of type A botulinum neurotoxin
Clostridial botulinum neurotoxins (BoNTs) cause neuroparalysis by blocking neurotransmitter release. The toxin's light chain (LC) acts as a Zn2+-endopeptidase. Type A botulinum nuerotoxin light chain (BoNT/A LC) was expressed in E. coli to high yield level. The recombinant BoNT/A LC, which retained the proteolytic activity and secondary structure, was used in this study. ^ During the translocation of the LC to the cytosol, it is exposed to the endosomal low pH. Low pH showed a dramatic change in the BoNT/A LC polypeptide folding as indicated by differential heat denaturation. Furthermore, binding of 1-anilinonaphthalenesulfonate revealed exposure of hydrophobic domains of BoNT/A LC at low pH. Low pH induced changes were completely reversible. Fluorescence measurements suggested that the two Trp residues are buried and constrained in a hydrophobic environment. The reversibility rules out a possible prerequisite of acid treatment of LC for an ultimate functional conformation. ^ BoNT/A is a zinc-endopeptidase that contains the consensus sequence HEXXH in LC. Substitution of Glu-224 in the motif with Gln (E224Q) resulted in a total loss of the endopeptidase activity, whereas substitution with Asp (E224D) retained ∼1% of the enzymatic activity. However, k m values for wild type and E224D LC were similar. Global structure, Zn2+ content, and substrate binding ability are retained in the mutants. Titration of Zn2+ to EDTA-treated wild-type and mutant proteins indicated similar Zn2+ binding affinity constants. These results suggest an essential and direct role of the carboxyl group of Glu-224 in the hydrolysis of the substrate. The location of the carboxyl group at a precise position is critical for the enzymatic activity. ^ BoNT contains one Zn2+/molecule. The active-site Zn 2+, which was easily displaced from the active site by ethylenediaminetetraacetate, reversibly binds to the LC in a stoichiometric manner. Enzymatic activity was completely abolished in zinc-depleted LC (apo-LC). However, Zn 2+ replenishment partially restored the activity in reZn2+ -LC. Comparable Km values in holo- and reZn2+-LC were observed, indicating a similar substrate binding ability. Removal of the zinc causes irreversible tertiary structural change while the secondary structure remains unchanged. Zinc-binding leads to enhanced thermal stability of LC, which is not identical in native holo-LC and reZn 2+-LC. ^
Li, Li, "Molecular basis of the endopeptidase activity of type A botulinum neurotoxin" (2000). Doctoral Dissertations Available from Proquest. AAI9978520.