The protease kallikrein 4 (KLK4) plays a pivotal role during dental enamel formation by degrading the major enamel protein, amelogenin, prior to the final steps of enamel hardening. KLK4 dysfunction is known to cause some types of developmental defect in enamel but the mechanisms responsible for transient retention of KLK4 in semi-hardened enamel matrix remain unclear. To address contradictory reports about the affinity of KLK4 for enamel hydroxyapatite-like mineral, we used pure components in quasi-physiological conditions and found that KLK4 binds hydroxyapatite directly. Hypothesising KLK4 self-destructs once amelogenin is degraded, biochemical analyses revealed that KLK4 progressively lost activity, became aggregated, and autofragmented when incubated without substrate in both the presence and absence of reducer. However, with non-ionic detergent present as proxy substrate, KLK4 remained active and intact throughout. These findings prompt a new mechanistic model and line of enquiry into the role of KLK4 in enamel hardening and malformation. •Contradictions about KLK4 binding to hydroxyapatite were resolved biochemically.•A proxy substrate stabilises KLK4 against inactivation, aggregation and autolysis.•A mechanistic model involving KLK4 retention and autodegradation is proposed.