Proteolytic fragments of insulysin (IDE) retain substrate binding but lose allosteric regulation

Treatment of an N-terminal-containing His₆-tagged insulysin (His₆-IDE) with proteinase K led to the initial cleavage of the His tag and linker region. This was followed by C-terminal cleavages resulting in intermediate fragments of ∼95 and ∼76 kDa and finally a relatively stable ∼56 kDa fragment. The ∼76 and ∼56 kDa fragments exhibited a low level of catalytic activity but retained the ability to bind the substrate with a similar affinity as the native enzyme. The kinetics of the reaction of the IDE ∼76 and ∼56 kDa proteolytic fragments with a synthetic fluorogenic substrate produced hyperbolic substrate versus velocity curves, rather than the sigmoidal curve obtained with His₆-IDE. The ∼76 and ∼56 kDa IDE proteolytic fragments were active toward the physiological peptides β-endorphin, insulin, and amyloid β peptide 1-40. Although activity was reduced by a factor of ∼10³-10⁴ with these substrates, the relative activity and the cleavage sites were unchanged. Both the ∼76 and ∼56 kDa fragments retained the regulatory cationic binding site that binds ATP. Thus, the two proteinase K cleavage fragments of IDE retain the substrate- and ATP-binding sites but have low catalytic activity and lose the allosteric kinetic behavior of IDE. These data suggest a role of the C-terminal region of IDE in allosteric regulation.