Abstract
Fatty acid amide hydrolase (FAAH) is a dimeric, membranebound enzyme that degrades neuromodulatory fatty acid amides and esters and is expressed in mammalian brain and peripheral tissues. The cleavage of approximately 30 amino acids from each subunit creates an FAAH variant that is soluble and homogeneous in detergent-containing buffers, opening the avenue to the in vitro mechanistic and structural studies. Here we have studied the stability of FAAH as a function of guanidinium hydrochloride concentration and of hydrostatic pressure. The unfolding transition was observed to be complex and required a fitting procedure based on a three-state process with a monomeric intermediate. The first transition was characterized by dimer dissociation, with a free energy change of approximately 11 kcal/mol that accounted for approximately 80% of the total stabilization energy. This process was also paralleled by a large change in the solvent-accessible surface area, because of the hydration occurring both at the dimeric interface and within the monomers. As a consequence, the isolated subunits were found to be much less stable (DeltaG approximately 3 kcal/mol). The addition of methoxyarachidonyl fluorophosphonate, an irreversible inhibitor of FAAH activity, enhanced the stability of the dimer by approximately 2 kcal/mol, toward denaturant- and pressure-induced unfolding. FAAH inhibition by methoxyarachidonyl fluorophosphonate also reduced the ability of the protein to bind to the membranes. These findings suggest that local conformational changes at the level of the active site might induce a tighter interaction between the subunits of FAAH, affecting the enzymatic activity and the interaction with membranes.
Highlights
Fatty acid amide hydrolase (FAAH) is a dimeric, membranebound enzyme that degrades neuromodulatory fatty acid amides and esters and is expressed in mammalian brain and peripheral tissues
These findings suggest that local conformational changes at the level of the active site might induce a tighter interaction between the subunits of FAAH, affecting the enzymatic activity and the interaction with membranes
Guanidinium hydrochloride (GdmHCl)-induced Unfolding of FAAH—The stability of FAAH has been studied by equilibrium unfolding measurements, following the change in intrinsic fluorescence
Summary
EFFECT OF THE INHIBITOR METHOXYARACHIDONYL FLUOROPHOSPHONATE ON THE CONFORMATION AND MEMBRANE BINDING OF FATTY ACID AMIDE HYDROLASE*. It is widely recognized that the biological activity of FAAs depends on the “metabolic control” of their endogenous tone [3], and in the last few years evidence has accumulated that points to FAAH as the key regulator of FAAs concentration in vivo [4, 5] This enzyme is becoming the subject of intense investigation, and the design of ad hoc inhibitors able to tune its catalytic activity is a hot spot in medicinal chemistry (for a recent review see Ref. 6). The data provide strong evidence that the protein quaternary structure is needed to achieve stability, by shielding from solvent the residues at the dimeric interface and those buried inside each subunit Not surprisingly, this folding strategy appears to be optimal to match the functional requirements of FAAH
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