Mechanism-based traps enable protease and hydrolase substrate discovery

Hydrolase enzymes, including proteases, are encoded by 2–3% of the genes in the human genome and 14% of these enzymes are active drug targets 1 . However, the activities and substrate specificities of many proteases—especially those embedded in membranes—and other hydrolases remain unknown. Here we report a strategy for creating mechanism-based, light-activated protease and hydrolase substrate traps in complex mixtures and live mammalian cells. The traps capture substrates of hydrolases, which normally use a serine or cysteine nucleophile. Replacing the catalytic nucleophile with genetically encoded 2,3-diaminopropionic acid allows the first step reaction to form an acyl-enzyme intermediate in which a substrate fragment is covalently linked to the enzyme through a stable amide bond 2 ; this enables stringent purification and identification of substrates. We identify new substrates for proteases, including an intramembrane mammalian rhomboid protease RHBDL4 (refs. 3 , 4 ). We demonstrate that RHBDL4 can shed luminal fragments of endoplasmic reticulum-resident type I transmembrane proteins to the extracellular space, as well as promoting non-canonical secretion of endogenous soluble endoplasmic reticulum-resident chaperones. We also discover that the putative serine hydrolase retinoblastoma binding protein 9 (ref. 5 ) is an aminopeptidase with a preference for removing aromatic amino acids in human cells. Our results exemplify a powerful paradigm for identifying the substrates and activities of hydrolase enzymes.