Clickable, photoreactive inhibitors to probe the active site microenvironment of fatty acid amide hydrolase

Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that degrades the endocannabinoid anandamide (AEA) and several other bioactive lipid amides. The catalytic mechanism of FAAH has been largely elucidated, and structural models of the enzyme suggest that it may recruit its hydrophobic substrates directly from the lipid bilayer of the cell. Testing this hypothesis, however, requires new tools to explore FAAH-substrate interactions in native cell membranes. Here, we have addressed this problem by creating clickable, photoreactive inhibitors that probe the microenvironment surrounding the FAAH active site. We show that these probes can be used directly in cell membranes, where distinct crosslinked adducts are observed for inhibitors that are buried within versus exposed to the external environment of the FAAH active site.