Probing the mechanistic role of glutamate residue in the zinc-binding motif of type A botulinum neurotoxin light chain

Type A botulinum neurotoxin (BoNT/A) is a zinc endopeptidase that contains the consensus sequence HEXXH (residues 223-227) in the toxic light chain (L C). 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 g lutamate is a crucial amino acid involved in catalysis. The functional impl ication of E224 in the motif of LC was investigated by replacing the residu e with glutamine and aspartate using site-directed mutagenesis. Substitutio n 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 (k(cat) 140 vs 1.9 min(-1), respectively). However, K-m values for wild-type and E224D BoNT/A LC were similar, 42 and 50 mu M, res pectively. Global structure, in terms of secondary structure content and to pography of aromatic amino residues, Zn2+ content, and substrate binding ab ility 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 th e carboxyl group of Glu-224 in the hydrolysis of the substrate. The locatio n 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.