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The solution structure of a 55-amino-acid Kunitz-type proteinase inhibitor, ShPI, purified from the Caribbean sea anemone Stichodactyla helianthus, was determined by NMR spectroscopy. Nearly complete sequence-specific 'H-NMR assignments were obtained at pH 4.6 and 36 OC, and stereospecific assignments were determined for 23 pairs of diastereotopic substituents. A data set of 666 upper distance limit constraints and 122 dihedral angle constraints collected on this basis was used as input for a structure calculation with the program DIANA. Following energy minimization with the program OPAL, the average root-mean-square diviation (RMSD) of the 20 DIANA conformers used to represent the solution structure relative to the mean structure is 61 pm for all backbone atoms N, Ca and C', and 106 pm for all heavy atoms of residues 2-53. This high-quality solution structure of ShPI has a nearly identical molecular architecture as the bovine pancreatic trypsin inhibitor (BPTI), despite a mere 35% of sequence similarity between the two proteins. Exchange rates measured for 48 out of the 51 backbone amide protons showed that the positions of 20 slowly exchanging amide protons correlate well with hydrogen bonds involving these protons in the energyminimized solution structure. The solution structure of ShPI is compared to the four homologous proteins for which the three-dimensional structure is also available.

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After screening 25 marine invertebrates, a novel metallocarboxypeptidase (SmCP) has been identified by activity and MS analytical approaches, and isolated from the marine annelid Sabellastarte magnifica. The enzyme, which is a minor component of the molecularly complex animal body, as shown by 2D gel electrophoresis, has been purified from crude extracts to homogeneity by affinity chromatography on potato carboxypeptidase inhibitor and by ion exchange chromatography. SmCP is a protease of 33792 Da, displaying N‐terminal and internal sequence homologies with M14 metallocarboxypeptidase‐like enzymes, as determined by MS and automated Edman degradation. The enzyme contains one atom of Zn per molecule, is activated by Ca2+ and is drastically inhibited by the metal chelator 1,10‐phenanthroline, as well as by excess Zn2+ or Cu2+, but moderately so by EDTA. SmCP is also strongly inhibited by specific inhibitors of metallocarboxypeptidases, such as benzylsuccinic acid and the protein inhibitors found in potato and leech (i.e. recombinant forms, both at nanomolar levels). The enzyme displays high peptidase efficiency towards pancreatic carboxypeptidase‐A synthetic substrates, such as those with hydrophobic residues at the C‐terminus but, remarkably, also towards the acidic ones. This property, previously described as for carboxypeptidase O‐like activity, has been shown on long peptide substrates by MS. The results obtained in the present study indicate that SmCP is a novel member of the M14 metallocarboxypeptidases family (assignable to the M14A or pancreatic‐like subfamily) with a wider specificity that has not been described previously.