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Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins
Published on: June 20, 2019
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The endocannabinoid hydrolase FAAH is an allosteric enzyme
Enrico Dainese1, Sergio Oddi2,3, Monica Simonetti4
1Faculty of Biosciences, and Technology for Food Agriculture and Environment, University of Teramo, Teramo, Italy. edainese@unite.it.
Scientific Reports
|February 12, 2020
Summary
Fatty acid amide hydrolase (FAAH) enzyme inhibition reveals allosteric communication between its subunits. A single mutation can disrupt this communication, offering new therapeutic targets for controlling endocannabinoid activity.
Area of Science:
- Biochemistry
- Enzymology
- Pharmacology
Background:
- Fatty acid amide hydrolase (FAAH) regulates endocannabinoids like N-arachidonoylethanolamine (AEA).
- FAAH functions as a homodimeric enzyme, with parallel monomer orientation potentially facilitating substrate binding and cleavage.
Purpose of the Study:
- To investigate the allosteric inhibition mechanism of human and rat FAAH.
- To explore the role of specific amino acid residues at the dimer interface in FAAH activity and inhibition.
Main Methods:
- Enzyme kinetics studies using wild-type and mutant FAAH (W445Y, F432A).
- Inhibition assays with enzyme inhibitors at varying stoichiometric ratios.
- Analysis of N-arachidonoylethanolamine (AEA) hydrolysis kinetics.
Main Results:
- Full inhibition of FAAH was achieved with a 1:1 homodimer:inhibitor ratio, indicating one active site occupation is sufficient for blockade.
- The W445Y mutation impaired full inhibition, while F432A showed reduced activity but maintained full inhibition at the 1:1 ratio.
- Kinetic analysis revealed an allosteric hydrolysis of AEA by human and rat FAAH (Hill coefficients ~1.9 and ~1.6, respectively), with W445Y showing a coefficient of ~1.0.
Conclusions:
- FAAH exhibits unprecedented allosterism, with communication between subunits potentially mediated by amino acid W445 at the dimer interface.
- These findings suggest a novel mechanism for controlling FAAH activity.
- The study provides a basis for designing new therapeutic molecules targeting FAAH for conditions involving endocannabinoid dysregulation.
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