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 (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 (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 microM, respectively. Global structure, in terms of secondary structure content and topography of aromatic amino residues, Zn(2+) content, and substrate binding ability are retained in the enzymatically inactive mutants. Titration of Zn(2+) to EDTA-treated wild-type and mutant proteins indicated identical enthalpy for Zn(2+) 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.
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Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, Dartmouth, Massachusetts 02747, USA.
Binz
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Niemann
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Singh
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D020816:Amino Acid Motifs / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D019274:Botulinum Toxins, Type A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004735:Energy Transfer; D004789:Enzyme Activation / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005973:Glutamine / Q000235:genetics; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity / Q000235:genetics; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism*
D005973:Glutamine; D001224:Aspartic Acid; D018698:Glutamic Acid; D008666:Metalloendopeptidases; D019274:Botulinum Toxins, Type A; D015032:Zinc