Closing the Gate to the Active Site

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 ≈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 ≈11 kcal/mol that accounted for ≈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 (⌬G ≈ 3 kcal/mol). The addition of methoxyarachidonyl fluorophosphonate, an irreversible inhibitor of FAAH activity, enhanced the stability of the dimer by ≈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.Fatty acid amide hydrolase (FAAH) 3 is a dimeric, integral membrane protein (1) that brings about the degradation of important fatty acid neurotransmitters, such as oleamide and anandamide (2). These fatty acid amides (FAAs), collectively termed "endocannabinoids," modulate several aspects of mammalian pathophysiology, both at central and peripheral level (3). It is now 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). Therefore, 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). In fact, FAAH inhibitors might represent next-generation therapeutics for the treatment of drug and alcohol abuse (7), anxiety (8), cancer (9), as well as neurodegenerative (10, 11) and vascular (12) diseases. In addition, interest toward FAAH was boosted by the fact that this enzyme is the first and, to date, the only characterized mammalian member of a large group, referred to as the "amidase signature" (AS) family (13). More than 100 members of this class have been reported in the literature, mostly from bacteria or fungi. Despite its quite remarkable size, struct...

Computational insights into function and inhibition of fatty acid amide hydrolase

“…4, upper panel). This process is regulated by a phenylalanine residue (Phe432) located at the boundary between the two cavities, which may act as a "dynamic paddle" [21,45].…”

Membrane lipids are key modulators of the endocannabinoid-hydrolase FAAH

“…The fluorescence spectrum of FAAH is very broad due to the presence of more than one emitting species [37]; however, increasing the concentration of POPC vesicles led to a relevant red-shift of the wavelength at the emission maximum (λ max from 343.5 to 346.0 nm; Figure 3). The fluorescence spectrum of FAAH is very broad due to the presence of more than one emitting species [37]; however, increasing the concentration of POPC vesicles led to a relevant red-shift of the wavelength at the emission maximum (λ max from 343.5 to 346.0 nm; Figure 3).…”

“…The catalytically active transmembrane domain-lacking ( TM) mutant of rat FAAH [1] was expressed with a His 6 tag in E. coli BL21(DE3)pLysS competent cells (Merck) using the pTrcHisAFAAH-TM plasmid [2] as reported previously [3]. Briefly, rat TM-FAAH cDNA was digested with the EcoRI and XhoI restriction enzymes, in phase with the coding sequence for the tail of six histidines, and was inserted into the pTrcHisA plasmid following a standard procedure (Invitrogen Life Technologies).…”

“…Briefly, rat TM-FAAH cDNA was digested with the EcoRI and XhoI restriction enzymes, in phase with the coding sequence for the tail of six histidines, and was inserted into the pTrcHisA plasmid following a standard procedure (Invitrogen Life Technologies). The column was pre-equilibrated with 10 volumes of 0.5 M Tris/HCl buffer (pH 7.5) containing 10 % (w/v) trehalose, 0.1 M NaCl and 10 mM imidazole (column buffer), and was then washed twice with the same buffer, but containing 20 mM imidazole, to remove the proteins bound unspecifically [3]. Transfected E. coli cells were pelleted and subjected to mechanical rupture in lysis buffer [10 % (w/v) trehalose, 0.5 % CHAPS, 0.1 M NaCl, 1 mg/ml lysozyme and 10 μl/ml protease inhibitor cocktail in 0.5 M Tris/HCl buffer (pH 7.5)].…”

Correlation of inhibitor effects on enzyme activity and thermal stability for the integral membrane protein fatty acid amide hydrolase

“…al. 9 studied the stability of FAAH as a function of guanidinium hydrochloride concentration and hydrostatic pressure and concluded that conformational changes mediated by inhibitor binding to the active site lead to tighter interaction between monomers and an increase in enzyme stability. This also resulted in a reduced ability of the protein to bind to membranes.…”