Probing the Mechanistic Role of Glutamate Residue in the Zinc-Binding Motif of Type A Botulinum Neurotoxin Light Chain

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Abstract
Type A botulinum neurotoxin (BoNT/A) is a zinc endopeptidase that contains the consensus sequence HEXXH (residues 223−227) in the toxic light chain (LC). The X-ray structure of the toxin has predicted that the two histidines of this motif are two of the three zinc-coordinating ligands and that the glutamate is a crucial amino acid involved in catalysis. The functional implication of E224 in the motif of LC was investigated by replacing the residue with glutamine and aspartate using site-directed mutagenesis. Substitution of Glu-224 with Gln (E224Q) resulted in a total loss of the endopeptidase activity, whereas substitution with Asp (E224D) retained about 1.4% of the enzymatic activity (kcat 140 vs 1.9 min-1, respectively). However, Km values for wild-type and E224D BoNT/A LC were similar, 42 and 50 μM, respectively. Global structure, in terms of secondary structure content and topography of aromatic amino residues, Zn2+ content, and substrate binding ability are retained in the enzymatically inactive mutants. Titration of Zn2+ to EDTA-treated wild-type and mutant proteins indicated identical enthalpy for Zn2+ binding. 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, as replacement of Glu-224 with Asp resulted in almost total loss of the activity.