Catalytic metzincin domain. (a) Catalytic endopeptidase subunit showing the peptide binding cleft and surrounding domains. The site binds six amino acids on either side of the bond to be hydrolyzed. Substrate is in an extended conformation and the bound amino acids are numbered (N- to C-terminus): …S³–S²–S¹–S^1′–S^2′–S^3′… The products are two peptides: …P³–P²–P¹–COOH and NH₂–P^1′–P^2′–P^3′… (Slightly modified from Fig. 1B of F.X. Gomis-Ruth, “Structural aspects of the metzincin clan of metalloendopeptidases.” Mol. Biotechnol. 24(2):157-202, 2003). (b) Active site crevice of the metzincin catalytic domain from above. The conserved zinc-binding motif has the structure: His¹⁷⁶-Glu¹⁷⁷-Xaa-Xaa-His¹⁸⁰-Xaa-Xaa-Gly-Xaa-Xaa-His¹⁸⁶. The numbering is for serralysin, an astacin family protein, but the relative positions of these amino acids within the motif are identical in all metzincins. The catalytic zinc ion (large sphere) has a coordinated water molecule (small sphere) whose hydrogen atoms are H-bonded to Glu¹⁷⁷ (bond shown in Fig. 8.2c). About 20 residues, downstream lies a tyrosine phenolic side chain that points back from beneath the active site as a result of a turn induced by the invariant methionine residue. The ligands involving tyrosine form a distorted trigonal bipyramid in which His¹⁷⁶, His¹⁸⁶, and the water molecule are equatorial. His¹⁸⁰ and Tyr²¹⁶, respectively, form the upper and lower axial ends (peaks of each pyramid). The Zn²⁺ ion is approximately 2.2 Å from the histidine ligands, 2 Å from the water molecule, and 2.8 Å from the phenolic O atom of Tyr²¹⁶. Adamalysins and matrilysins (and some other metzincins) have proline instead of tyrosine at the turn. In these enzymes, a cysteine residue within the pro-domain blocks the cleft by binding to the catalytic zinc ion and excluding water molecule binding (see text) (Modified from Park, H.I. and Ming, L.J. “Mechanistic studies of the astacin-like Serratia metalloendopeptidase serralysin: highly active (>2,000%) Co(II) and Cu(II) derivatives for further corroboration of a “metallotriad” mechanism.” J. Biol. Inorg. Chem. 7(6):600–610. 2002). (c) Mechanism of catalysis. (1) The tyrosine residue moves away from the zinc ion and the water molecule (green) attacks the substrate polypeptide (red) under the influence of the deprotonated glutamate residue. (2) The C-terminal peptide amino group picks up the proton and is released. (3) The remaining hydroxide anion attacks the carboxyl group, which remains held by the zinc ion but it is quickly replaced by another water molecule (Modified from University of Tours, France Web site: http://delphi.phys.univ-tours.fr/Prolysis/introprotease.html)

Architecture of astacins, adamalysin type-2, and matrix metalloproteases. Topology scheme of astacins, adamalysin type-2, and matrix metalloproteinases. The α-helices are shown as rods, β-sheet strands as arrows, and unstructured regions as thin lines. Key amino acids in the catalytic, zinc-binding motif are shown (white in black ellipse). Distinguishing features of each structure are shown as lighter gray for amino acids (black letter), and as unlabeled, secondary-structure elements. In addition to the catalytic zinc ion in all three structures, the calcium ion in adamalysin type-2, and the two calcium ions and second zinc ion in the matrix metalloproteinases (Abbreviated from Fig. 3 in F.X. Gomis-Ruth, “Structural aspects of the metzincin clan of metalloendopeptidases.” Mol. Biotechnol. 24(2):157–202, 2003)