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 targets¹. 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 bond²; 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.