Substrates incorporating unnatural amino acids are separately detectable for chymotrypsin and subtilisin with similar specificity

Protease specificity depends on the three-dimensional structure and sequence of the protease, and each protease plays a different role. Chymotrypsin and subtilisin have similar specificity for recognizing aromatic and hydrophobic amino acid residues. Previous methods using natural amino acids have difficulty in separately detecting proteases with similar specificity. To overcome this, we developed an effective substrate incorporating unnatural amino acids using a simple method that changes only one residue of a substrate. Various 7-amino-4methylcoumarin (AMC) peptide substrates were synthesized in terms of hydrophobicity, aromaticity, and conformation, including unnatural amino acids 2-amino-4-ethylhexanoic acid (AEH) and cyclohexylalanine (Cha). Although AEH substrate is highly hydrophobic, steric hindrance based on the branched side chain of AEH residue reduced the recognition of the substrate by the two proteases. Cha substrate with a six-membered ring side chain was most preferably recognized by chymotrypsin but was slightly recognized by subtilisin. This shows that Cha substrate can separately detect the two proteases with similar specificity. Therefore, these results indicate that protease with similar specificities is separately detectable by substrates with a side chain having structural degrees of freedom that provide different steric hindrance.