Figure 1. Positional specificity of protease activity. Proteases can be divided into exopeptidases and endopeptidases. The exopeptidases act only near the ends of polypeptide chains. Those acting at a free N-terminus may liberate a single amino acid residue (aminopeptidases), a dipeptide (dipeptidyl-peptidases) or a tripeptide (tripeptidyl-peptidases). Those acting at a free C-terminus liberate a single residue (carboxypeptidases) or a dipeptide (peptidyldipeptidases). Other exopeptidases are specific for dipeptides (dipeptidases), or remove terminal residues that are substituted, cyclized or linked by isopeptide bonds (peptide linkages other than those of -carboxyl to -amino groups) ( peptidases). In the figure, the circles represent amino acid residues, the down-arrows show the bonds that are hydrolysed, and the brackets attached to the arrows indicate the part of the substrate molecule typically recognized by the specificity sites of the enzymes prior to catalysis, and thus directing specificity. Proteases that act internally in polypeptide chains (usually whole protein molecules) are called endopeptidases. Primary determinants of endopeptidase specificity are amino acids near the scissile peptide bond on either side.

Figure 2. Scheme for the specificity subsites of proteases that dictate their sequence specificity. It can be seen that the specificity subsites are numbered from the catalytic site, S1, S2, , Sn towards the N-terminus of the substrate, and S1¢,S2¢, , Sn¢ towards the C-terminus. The amino acids they accommodate are numbered P1, P2, , Pn and P1¢, P2¢, , Pn¢, respectively.