X-ray absorption spectroscopy study of zinc coordination in tetanus neurotoxin, astacin, alkaline protease and thermolysin

Silvia Morante, Lars Furenlid, Giampietro Schiavo, Fiorella Tonello, Robert Zwilling, Cesare Montecucco

Research output: Contribution to journalArticlepeer-review

17 Scopus citations


Tetanus and botulinum neurotoxins constitute a new group of Zn-endopeptidases which has recently been actively investigated with the purpose of correlating their biochemical properties Io their neurobiocytosis inhibitory capacity. Crystallographic data show that Zn-endopeptidases are characterized by an active site with a Zn atom coordinated to two histidines and a glutamate-bound waler molecule. The two histidines and the glutamate residues belong to the HEXXH motif which is characteristic of most Zn-endopeptidases. A fourth metal ligand is a glutamate in thermolysin-like proteinases, hut it is an histidine in the astacin family of proteinases and in alkaline protease. Astacin and alkaline protease possess a tyrosine as fifth Zn ligand, whose position in the case of alkaline protease could not be determined by X-ray crystallography. Not much is known about the atom arrangement around the active site in tetanus neurotoxin. In this work X-ray absorption spectroscopy has been used to obtain information on the Zn coordination mode in tetanus neurotoxin. The near-edge and extended fine-structure absorption spectra of this toxin are compared with those of astacin, alkaline protease and thermolysin. The present data and sequence information suggest a new pattern of Zn coordination in tetanus neurotoxin with one wuter molecule and three aromatic residues as metal ligands. These residues are the two histidines of the characteristic motif and a tyrosine which is tentatively identified with Tyr242, on the basis of sequence comparison and mutagenesis experiments. The mean distances of the Zn from the nearest coordinated atoms is reported. Our results indicate that alkaline protease, like astacin, also possesses a tyrosine as a fifth ligand.

Original languageEnglish (US)
Pages (from-to)606-612
Number of pages7
JournalEuropean Journal of Biochemistry
Issue number3
StatePublished - 1996


  • Extended X-ray absorption fine structure
  • X-ray absorption spectroscopy
  • Zinc-endopeptidases

ASJC Scopus subject areas

  • Biochemistry


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