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Introduction

Fatty acid amide hydrolase (FAAH) degrades fatty acid amides (FAAs) to terminate their signaling activity ^[1]. A serine hydrolase from the Amidase signature superfamily of enzymes (other amidases), FAAH degrades endocannabinoid signaling lipids, molecules associated with pain relief ^[2]. Because endocannabinoids are lipid molecules, they cannot be compartmentalized in vesicles (the degradation method for other neurotransmitters) and must instead be degraded in the bilayer of the cell membrane. FAAH is an integral membrane protein that degrades FAAs as they enter the membrane bilayer, allowing the cell to terminate the activity of signaling molecules that cannot be contained within a vesicle for degredation ^[1]. Current FAAH research aims to find inhibitors for the enzyme, which would prolong the pain alleviation provided by endocannabinoid molecules ^[2].

Figure 1: FAAH Monomer Subunit; Enzyme in teal, Ligand in pink. FAAH Crystal Structure.

Hydrolase Information

Crystal structures of FAAH show that the enzyme is a (monomers in different colors) in solution, with each subunit having a mass of 63 kD ^[1]. The protein's of 11 strands (blue) is surrounded by 24 (green). The enzyme is embedded in the cell membrane to catch the lipid signaling molecules that can diffuse through membranes; the hydrolase has a membrane binding cap, a consisting of alpha helices 18 and 19. These helices present hydrophobic amino acid residues that help FAAH interact with the hydrophobic region of the lipid bilayer ^[1]. Specifically, (black) interact with the hydrophobic region of the membrane to anchor the enzyme in the lipid bilayer. The FAAH structure shows an entry channel leading from the lipid bilayer to the enzyme's active site, providing a path for endocannabinoids to enter the hydrolase. This entry channel is amphipathic to accommodate the entire lipid signaling molecule. Hydrophobic amino acid residues interact with the lipid signaling molecules' nonpolar tails, while charged R486 and D403 residues in the entry channel accommodate the polar head groups. In addition, FAAH possesses an exit channel leading from the active site to the cell's cytoplasm, allowing the release of polar compounds released from lipid cleavage and the entry of water molecules necessary for the FAAH mechanism to proceed ^[1]. Different inhibitors have been designed to learn more about species selectivity ^[3] and binding flexibility ^[4] in FAAH. For example, the (red) is the inhibitor used on a humanized rat FAAH protein. Although PF-750 showed preference for human FAAH, rat FAAH is easier to express; this demonstrates species selectivity of inhibitors ^[3].

Catalytic Triad

Mutagenesis and inhibitor studies have shown that FAAH has a , consisting of Ser241, Ser217, and Lys142 ^[1]. Ser-Ser-Lys catalytic triads are not often seen in hydrolases, making FAAH an enzyme of interest for additional research to better determine how proteins with this catalytic triad function. Ser241 acts as the catalytic nucleophile for the cleavage of amide bonds (Figure 2) ^[1]. Inhibitors are able to inactivate the catalytic triad by providing a substrate containing a leaving group, such as aniline, that is a more favorable leaving group than the Ser241 hydroxyl group. With the serine bound to the carbonyl carbon, FAAH is no longer able to accommodate any more substrates ^[3]. FAAH also requires two water molecules in its active site to properly position and cleave amide bonds. One water molecule (W1) deacylates the substrate, and the other (W2) helps coordinate W1 through the catalytic K142 (Figure 3) ^[5] .

Figure 2: Catalytic Triad and PF-750 Inhibitor Mechanism; The combination of Ser241, Ser217, and Lys142 provides a partial negative charge on the Ser241 hydroxyl group. The Ser241 oxygen attacks the carbonyl carbon of PF-750, resulting in a tetrahedral intermediate. Aniline is released as a leaving group, with the remaining portion of PF-750 still covalently bound to Ser241. FAAH is now inactivated, unable to accommodate any new ligands ^[3].

Relationship to other proteins

The hydrolytic water molecules important to FAAH's function suggest an evolutionary relationship of this hydrolase to other enzymes. The structures of other serine hydrolases also display a catalytic water molecule in their active sites. Because hydrolases that are non-homologous to FAAH also require a water molecule to cleave bonds, a functional convergance has inferred between amidase signature enzymes (such as FAAH) and other classes of serine proteases ^[5].

This evidence of convergent evolution between FAAH and other amidase signature enzymes supports the Bürgi-Dunitz theory. The Bürgi-Dunitz theory proposes that nucleophiles tend to follow a specific trajectory when attacking a carbonyl, resulting in many enzyme mechanisms having the same angle between an incoming nucleophile and the carbonyl it attacks. Water molecules in the active sites of enzymes are specifically positioned to force the nucleophile to approach at the exact "Bürgi-Dunitz angle" of 107°. The determination that FAAH also has water molecules in its active site, helping the nucleophile to attack the amide carbonyl at a specific angle, adds additional support to the Bürgi-Dunitz theory ^[5].