title: Role of zinc binding in type A botulinum neurotoxin light chain's toxic structure.
abstract: Clostridial neurotoxins are zinc endopeptidases, and each contains one Zn(2+)/molecule. To investigate the structural/functional role of Zn(2+) in botulinum neurotoxin light chain (the enzymatic subunit of the neurotoxin), the effect of the removal of zinc on protein folding and enzyme kinetics was investigated. The active site Zn(2+), which was easily displaced from the active site by ethylenediaminetetraacetate, reversibly binds to the BoNT/A light chain (LC) in a stoichiometric manner. Enzymatic activity was completely abolished in the zinc-depleted light chain (apo-LC). However, Zn(2+) replenishment partially restored the activity in the re-Zn(2+)-LC (k(cat) = 72 min(-)(1)) compared to the holo-LC (k(cat) = 140 min(-)(1)). Comparable K(m) values in the holo- and re-Zn(2+)-LC were observed (41 and 55 microM, respectively), indicating a similar substrate binding ability. We investigated the structural basis of a 3-fold difference in the catalytic efficiency of the native holo-LC and re-Zn(2+)-LC by analyzing secondary and tertiary structural parameters. Removal of the zinc causes irreversible tertiary structural change while the secondary structure remains unchanged. Zinc binding leads to enhanced thermal stability of the LC, which is not identical in the native holo-LC and re-Zn(2+)-LC.
authors.affiliation: Department of Chemistry and Biochemistry and Center for Marine Science and Technology, University of Massachusetts Dartmouth, Dartmouth, Massachusetts 02747, USA.
authors.forename: L
authors.initials: L
authors.lastname: Li
authors.forename: B R
authors.initials: BR
authors.lastname: Singh
mesh_terms: D001665:Binding Sites; D019274:Botulinum Toxins, Type A / Q000737:chemistry* / Q000378:metabolism* / Q000633:toxicity; D002942:Circular Dichroism; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D015032:Zinc / Q000378:metabolism*
chemical_list: D010450:Endopeptidases; D019274:Botulinum Toxins, Type A; D015032:Zinc