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Zinc metallopeptidases that contain the His-Glu-Xaa-Xaa-His (HEXXH) motif generally have a third ligand of the metal ion that may be either a Glu residue (in clan MA) or a His residue (in clan MB) (Rawlings and Barrett (1995) Methods Enzymol. 248, 183–228). Thimet oligopeptidase has not yet been assigned to either clan, and both Glu and His residues have been proposed as the third ligand. We mutated candidate ligand residues in the recombinant enzyme and identified Glu, His and Asp residues that are important for catalytic activity and/or stability of the protein. However, neither of the Glu and His residues close to the HEXXH motif that have previously been suggested to be ligands is required for the binding of zinc. We conclude that thimet oligopeptidase is not a member of clan MA or clan MB and it is likely that the enzyme possesses a catalytic site and protein fold different from those identified in any metallopeptidase to date. The definitive identification of the third zinc ligand may well require the determination of the crystallographic structure of thimet oligopeptidase or one of its homologues.

We show by site-directed mutagenesis that the catalytic residues of mammalian legumain, a recently discovered lysosomal asparaginycysteine endopeptidase, form a catalytic dyad in the motif His-Gly-spacer-Ala-Cys. We note that the same motif is present in the caspases, aspartate-specific endopeptidases central to the process of apoptosis in animal cells, and also in the families of clostripain and gingipain which are arginyl/lysyl endopeptidases of pathogenic bacteria. We propose that the four families have similar protein folds, are evolutionarily related in clan CD, and have common characteristics including substrate specificities dominated by the interactions of the S1 subsite.

The amino acid sequence of papaya proteinase IV (PPIV), a major proteinase from the latex of Carica papaya [(1989) Biochem. J. 261, 469-476] is described. The enzyme has a high degree of sequence identity with papaya proteinase III, chymopapain and papain (81, 70 and 67%, respectively), and is clearly a member of the papain superfamily of cysteine proteinases. Nevertheless, the sequence shows substitution of certain residues conserved in all other known members of the superfamily. It is suggested that some of these substitutions may account for the unusual specificity of PPIV.

The amino acid sequence of stem bromelain, the major cysteine proteinase from pineapple stem is described. It shows that the enzyme is a member of the papain superfamily of cysteine proteinases, but is not very closely related to any other known member of this group. The sequence shows mutation or deletion of several residues that have been conserved in cysteine proteinases examined previously, including Asn-175 (papain). We suggest that some of these changes have the effect of altering the active-site geometry of stem bromelain, and that this accounts for the resistance of the enzyme to inhibition by cystatins and E-64 [L-3-carboxy-2,3-trans-epoxypropionylleucylamido(4-guanidino)butane].