Interdependency of sequence and positional specificities for cysteine proteases of the papain family

The specificity of cysteine proteases is characterized by the nature of the amino acid sequence recognized by the enzymes (sequence specificity) as we ll as by the position of the scissile peptide bond (positional specificity, i.e., endopeptidase, aminopeptidase, or carboxypeptidase). In this paper, the interdependency of sequence and positional specificities for selected m embers of this class of enzymes has been investigated using fluorogenic sub strates where both the position of the cleavable peptide bond and the natur e of the sequence of residues in P-2-P-1 are varied. The results show that cathepsins K and L and papain, typically considered to act strictly as endo peptidases, can also display dipeptidyl carboxypeptidase activity against t he substrate Abz-FRF(4NO(2))A and dipeptidyl aminopeptidase activity agains t FR-MCA. In some cases the activity is even equal to or greater than that observed with cathepsin B and DPP-I (dipeptidyl peptidase I), which have be en characterized previously as exopeptidases. In contrast, the exopeptidase activities of cathepsins K and L and papain are extremely low when the P-2 -P-1 residues are A-A, indicating that, as observed for the normal endopept idase activity, the exopeptidase activities rely heavily on interactions in subsite S-2 (and possibly S-1). However, cathepsin B and DPP-I are able to hydrolyze substrates through the exopeptidase route even in absence of pre ferred interactions in subsites S-2 and S-1. This is attributed to the pres ence in cathepsin B and DPP-I of specific structural elements which serve a s an anchor for the C- or N-terminus of a substrate, thereby allowing favor able enzyme-substrate interaction independently of the P2-P1 sequence. As a consequence, the nature of the residue at position P-2 of a substrate, whi ch is usually the main factor determining the specificity for cysteine prot eases of the papain family, does not have the same contribution for the exo peptidase activities of cathepsin B and DPP-I.