Molecular Mechanism of FAAH Substrate Recruitment

Scripps Research Institute, La Jolla CA

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Abstract

DESCRIPTION (provided by applicant): There are a number of integral membrane proteins involved in key biological processes, including several cyclooxygenases, which share the common structural feature of a hydrophobic plateau region that is heorized to anchor the enzymes to cellular membranes. All of these enzymes have very lipophilic substrates, leading to the current theory that the opening to the active site resides near this membrane anchor, allowing these enzymes to access their substrates directly from the membrane. We propose to synthesize bifunctional lipophilic probes to target a representative enzyme defined by this structural feature, fatty acid am/de hydrolase. The probes are designed with an irreversible affinity label on one end to target FAAH's active site and a photoactivatable crosslinking group at the other end. Varying the alkyl chain length separating these reactive groups should allow detailed mapping of the binding pocket with the longer chain agents designed to access the chemical environment just outside of the active site. If these probes crosslink FAAH's active site to lipid constituents of the membrane, this would provide the first biochemical evidence that the molecular mechanism of substrate recruitment is directly from the lipid bilayer.