Molecular Bases for Recognition of Short Peptides and Cysteine‐Directed Modifications of Insulin‐Degrading Enzyme

Abstract

Insulin degrading enzyme (IDE) utilizes a catalytic chamber to cleave substrates like insulin and amyloid beta. Interactions with substrates occur at an exosite ~30 A away from the catalytic center that anchors the N‐term of substrates to facilitate binding and cleavages at the catalytic site. However, IDE also degrades peptide substrates that are too short to occupy both the catalytic and the exosite simultaneously. Here, we use kinins to address the kinetics and regulation of IDE with short peptides. IDE specifically degrades kinins at the Pro/Phe site. A 1.9 A structure of bradykinin‐bound IDE reveals the binding of bradykinin only to the exosite and not the catalytic site. This is in agreement with the high Km of bradykinin for IDE. We also found that human IDE is potently inhibited by physiologically relevant concentrations of S‐nitrosylation and oxidation agents. Cysteine‐directed modifications play a key role, since an IDE mutant devoid of all 13 Cys is insensitive to the inhibition by these reagents. Specifically, cysteine 819 of human IDE is located inside the catalytic chamber pointing toward an extended hydrophobic pocket and is critical for the inactivation. Thiol‐directed modification of this residue likely causes local structural perturbation to reduce substrate binding and catalysis.