Zinc protease inhibitors binding

Figure 7. The structure of thermolysin. Ribbon representation of the structure of thermolysin (silver, Brookhaven Databank [53] code 3TLN) shown with a bound inhibitor (green). The catalytic zinc atom (cyan) and structural calcium atoms (magenta) are shown. The active site is located between the N-terminal zinc protease domain and the alpha helical C-terminal domain. Zinc binding residues are in blue and the residue assisting catalysis is shown in red.

The most conserved segment of the L chain of CNTs is a central region that contains a His-Glu-Xaa-Xaa-His zinc-binding motif characteristic of zinc-endopeptidases, thus suggesting that TeTx and the BoNTs may inhibit neuroexocytosis through a zinc-endopeptidase activity. This hypothesis was confirmed with two experimental approaches in Aply-sia neurons. First, the lack of toxicity of the apo-TeTx L chain demonstrated the essential role of the metal atom in toxin activity (Schiavo et ai, 1992 a, b). Second, phosphoramidon, a very specific inhibitor of zinc-endopeptidases, was shown to inhibit TeTx-induced blockade of ACh release (Schiavo ef ai, 1992 b). These results were the first clear evidence that the L chain of TeTx was acting via a metallo-protease activity. 

Thermolysin (TEN EC 3.4.24.28), a thermostable bacterial protease isolated from Bacillus thermoproteolyticus, has been studied as the prototype of zinc-metallopeptidases at a time where no crystal structure was available for this class of proteases [122]. Crystallographic analysis of a number of TLN/inhibitor complexes has allowed an understanding of the binding mode of these inhibitors and allowed the mechanism of action of this protease to be determined [122]. These seminal studies have greatly inspired the development of NEP inhibitors, given the close stmctural relationship between TEN and NEP [123]. To examine further the structural relationships between these two peptidases, various phosphinic peptides were prepared. One of these compounds (58, Table 1) exhibits a Ki value of 26 nM toward thermolysin and 22 nM toward NEP [124]. 

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