Astacin, a digestive zinc-endopeptidase from the crayfish Astacus astacus L., is the prototype for the 'astacin family', which includes mammalian metallo-endopeptidases and developmentally regulated proteins of man, fruitfly, frog and sea urchin. Here we report the X-ray crystal structure of astacin, which reveals a deep active-site cleft, with the zinc at its bottom ligated by three histidines, a water molecule and a more remote tyrosine. The third histidine (His 102) forms part of a consensus sequence, shared not only by the members of the astacin family, but also by otherwise sequentially unrelated proteinases, such as vertebrate collagenases. It may therefore represent the elusive 'third' zinc ligand in these enzymes. The amino terminus of astacin is buried forming an internal salt-bridge with Glu 103, adjacent to His 102. Astacin pro-forms extended at the N terminus, as observed for some 'latent' mammalian astacin homologues, did not exhibit this 'active' conformation, indicating an activation mechanism reminiscent of trypsin-like serine proteinases.

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Astacin, a zinc-endopeptidase from the crayfish Astacus astucus L., represents a structurally distinct group of metalloproteinases termed the 'astacin family'. This protein family includes oligomeric membrane-bound proteins with zinc proteinase domains found in rodent kidneys (meprins A and B) and human small intestine (N-benzoyl-~-tyrosyl-4-aminobenzoate hydrolase). Another branch of this family comprises morphogenetically active proteins, which induce bone formation (human bone morphogenetic protein 1), or which play specific roles during the embryonic development of amphibians, fishes, echinoderms, and insects.The X-ray crystal structure of astacin has recently been solved to a resolution of 0.18 nm [Bode et al. (1992) Nature 358, 164-1671. This structure is different from hitherto known metalloendopeptidase structures and has been used in the present study to analyze the structures of the other members of the astacin protein family.Computer-assisted modelling of the proteolytic domain of the a-subunit of meprin A based on the astacin structure is possible if five single and one double residue deletions and three single residue insertions are implied. The proteinase domains of the other astacins can be included in the model-based sequence alignment by introducing additionally three insertions and one deletion. All of these insertions and deletions are observed in loop segments connecting regular secondary structure elements and should leave the overall structure unaltered.The topology of residues forming the zinc-binding active site of astacin corresponds to almost identical arrangements in all other astacins, suggesting that these are likewise metalloproteinases. Based on this similarity, it is proposed that the active-site metal ion of the astacins is penta-coordinated by three histidine residues, a tyrosine residue and a water molecule in a trigonal bipyramidal geometry. Other remarkable common features are a hydrophobic cluster in the N-terminal domain and a conserved, solvent-filled cavity buried in the C-terminal domain. Most interestingly, the amino-termini of all astacins can be modelled to start in a corresponding internal water cavity as seen in the astacin template, where the terminal alanine residue forms a water-linked salt bridge to Glu103, directly adjacent to Hisl02, the third zinc ligand. Therefore, an activation mechanism for the astacins reminiscent of that of the trypsin-like proteinases had been suggested, which now seems to be probable also for the other astacins.Besides these common traits, there are some minor differences which may have important consequences on the function of the astacins. A striking example are variations in the presumed S: substrate-binding site, which binds the amino acid side chain on the C-terminal side of the scissile bond of the substrate. In this subsite the crayfish proteinase astacin prefers short, uncharged residues. By contrast, meprin A accepts bulky, charged side chains in this position. This difference presumably can be explained by both the replacemen...

For the first time, the site of biosynthesis of a well characterized invertebrate digestive enzyme is localized. The enzyme chosen, Astacus protease, is a zinc-metalloenzyme occuring in high concentration in the gastric fluid of the freshwater crayfish Astacus astacus. Enzyme production was stimulated in adult crayfish either by feeding or by removal of the gastric fluid. Immunohistochemistry, cytology and investigation with radioactive tracers demonstrate that in the hours following stimulation, new enzyme was produced in the F-cells of the midgut gland and subsequently discharged into the midgut gland lumen. The enzyme was then accumulated and stored extracellularly in the cardiac stomach in active form. The mechanism of enzyme production observed in Astacus differs considerably from vertebrates suggesting an alternative model for synthesis and storage of digestive enzymes.

The amino acid sequence of a protease from the crayfish Astacus fluviatilis has been determined from overlapping sets of peptides derived largely by cleavage at Met, Lys, or Arg residues. The protein comprises 200 amino acid residues in a single polypeptide chain, corresponding to a molecular mass of 22,614 daltons. Two disulfide bonds link Cys-42 to Cys-198 and Cys-64 to Cys-84. The sequence of this invertebrate protease appears to be unique since it has no homologous relationship to any of the known protein sequences.