Each domain contributes a catalytic Asp residue, with an extended active site cleft localized between the two lobes of the molecule.

In modern-day enzymes, although the three-dimensional structures are very similar, the amino acid sequences are more divergent, except for the catalytic site motif, which is very conserved.

[6][7] One aspartate activates the water by abstracting a proton, enabling the water to perform a nucleophilic attack on the carbonyl carbon of the substrate scissile bond, generating a tetrahedral oxyanion intermediate stabilized by hydrogen-bonding with the second aspartic acid.

[1] Five superfamilies (clans) of aspartic proteases are known, each representing an independent evolution of the same active site and mechanisms.

This hydrogen bond stabilises the propeptide conformation and is probably responsible for triggering the conversion of pepsinogen to pepsin under acidic conditions.