Isoaspartate residues dramatically influence substrate recognition and turnover by proteases.

Posttranslational modifications influence the structure, stability and biological activity of proteins. Most of the reactions are enzyme-catalyzed, but some, such as asparagine (Asn) and glutamine (Gln) deamidation and the isoaspartate (isoAsp) formation within peptide chains, occur spontaneously. It has been previously shown that certain peptide sequences form isoAsp quite fast if the Asp stretches are exposed to the protein surface, thereby potentially changing susceptibility to proteolysis at these sites. This tempted us to investigate the activity of exo- and endopeptidases against Asp- or isoAsp-containing substrates. Members of the prolyl oligopeptidase family were unable to cleave substrates after proline if isoAsp was placed in the P2-position. Caspases, usually accepting Asp at P1-position of their substrates, did not cleave isoAsp-containing sequences. Similarly, the metal-dependent aminopeptidase amino peptidase N did not turnover N-terminal isoAsp-containing substrates, nor could the endopeptidase matrix metalloproteinase 3 (MMP 3) hydrolyze a serum amyloid A protein-like substrate if the sequence contained isoAsp instead of Asp. Also, the highly specific enterokinase, usually clipping after a stretch of four Asp residues and a lysine in the P1 position, could not turnover substrates if the P2 amino acid was replaced by isoAsp. In contrast, acylamino acid-releasing enzyme and dipeptidyl peptidases 1, 2 and 4 hydrolyzed substrates containing the isoAsp-Ala motif.