Sandbox Reserved 911: Difference between revisions

Crystal structures of FAAH show that the enzyme is a <scene name='57/573125/2vya/9'>homodimer</scene> (monomers in different colors) in solution, with each subunit having a mass of 63 kD <ref name="1MT5"/>. The protein's <scene name='57/573125/2vya/11'>twisted Beta sheet core</scene> of 11 strands (blue) is surrounded by 24 <scene name='57/573125/2vya/12'>alpha helices</scene> (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 <scene name='57/573125/2vya/14'>helix-turn-helix motif</scene> 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 <ref name="1MT5"/>.  Specifically, <scene name='57/573125/2vya/13'>alpha helix 18's hydrophobic residues</scene> (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 <scene name='57/573125/2vya/16'>entry channel</scene> is amphipathic to accommodate the entire lipid signaling molecule. Hydrophobic amino acid residues (green) interact with the lipid signaling molecules' nonpolar tails, while charged R486 and D403 residues (black) in the entry channel accommodate the polar head groups. In addition, FAAH possesses an [http://lem.ch.unito.it/didattica/infochimica/2008_Cioccolato/immagini/strutturafaah.jpg 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 <ref name="1MT5"/>. Different inhibitors have been designed to learn more about species selectivity <ref name="2VYA">PMID:18753625</ref> and binding flexibility <ref>PMID:19722626</ref> in FAAH. For example, the <scene name='57/573125/2vya/3'>PF-750 inhibitor</scene> (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 <ref name="2VYA"/>.

Mutagenesis and inhibitor studies have shown that FAAH has a <scene name='57/573125/2vya/15'>Ser-Ser-Lys catalytic triad</scene>, consisting of Ser241, Ser217, and Lys142 <ref name="1MT5"/>. 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) <ref name="1MT5"/>. [http://proteopedia.org/wiki/index.php/Fatty_acid_amide_hydrolase 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 <ref name="2VYA"/>. 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) <ref name="3LJ6">PMID:20493882</ref> .

[[Image:Catalytic_triad2.png|1000px|left|thumb|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 <ref name="2VYA"/>. ]]

The hydrolytic water molecules important to FAAH's function suggest an evolutionary relationship of this hydrolase to other enzymes. The structures of other [http://en.wikipedia.org/wiki/Serine_hydrolase 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 [http://en.wikipedia.org/wiki/Serine_protease serine proteases] <ref name="3LJ6"/>.

[[Image:3LJ6_Image4.png|400 px|left|thumb|Figure3: FAAH catalytic site with water molecules bound; Protein in green, Water molecules as red spheres, Measurements in orange]]

This evidence of convergent evolution between FAAH and other amidase signature enzymes supports the [http://euch6f.chem.emory.edu/burgidunitz.html 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 [http://www.nature.com/nrd/journal/v11/n4/images/nrd3673-f4.jpg (FAAH bound to inhibitor)]. Water molecules in the active sites of enzymes are specifically positioned to force the nucleophile to approach at the exact [http://3.bp.blogspot.com/-NvsQyVPnLIw/UO91-BQTVgI/AAAAAAAAExw/-seGZcjU3DE/s400/burgi-duntz+trajectory.png "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 <ref name="3LJ6"/>.  

The human nervous system has several types of chemical messengers, including amino acids, lipids, peptide hormones, and [http://en.wikipedia.org/wiki/Monoamine_neurotransmitter monoamines] <ref name="1MT5"/>. FAAH primarily [http://www.scripps.edu/cravatt/images/faah_cartoon.gif degrades] [http://www.chm.bris.ac.uk/motm/anandamide/ananh.htm anandamide] (AEA), a naturally-occurring signaling lipid that functions in the brain. AEA brings [http://www.painresearchforum.org/news/11548-flat-tening-pain pain relief] to the body. Inhibiting FAAH would likely sustain AEA signaling, leading to prolonged pain relief and decreased inflammation <ref name="2WAP"/>.

FAAH plays a role in endocannabinoid signaling that has intriguing potential as a drug target. This signaling system consists of endocannabinoid ligands (such as AEA), two G protein-coupled receptors (CB1 and CB2), and the enzymes that synthesize and degrade (such as FAAH) the signaling lipids. Previous research has explored the potential of regulating endocannabinoid signaling through the CB1 and CB2 receptors. However, molecules found to activate these receptors (such as [http://www.ch.ic.ac.uk/vchemlib/mim/bristol/thc/thc_text.htm tetrahydrocannabinol] (THC), the main psychoactive ingredient of [http://en.wikipedia.org/wiki/Cannabis_(drug) marijuana]), while providing the intended pain relief, also produce many undesirable side effects, such as decreased cognition and motor control. On the other hand, research involving FAAH inhibitors has shown that blocking this part of the pathway reduces pain without the unwanted side effects seen through CB1/CB2 activation. Thus, exploring the possibility of using FAAH inhibition to decrease pain relief with minimal side effects could lead to new pain treatment solutions. For example, the [http://drugdiscoveryopinion.com/2009/04/pf-3845-%E2%80%93-a-potent-and-selective-faah-inhibitor/ PF-3845 inhibitor]of FAAH is extremely selective for the enzyme and raises AEA levels for a prolonged duration of time. Therefore, this inhibitor could be explored as a possible pharmaceutical product to target FAAH when extended pain relief is desired <ref name="2WAP"/>.