Substrate Report for: o-NTFNAC

Acetylcholinesterase (AChE) exhibits two different activities, namely esterase and aryl acylamidase (AAA). Unlike esterase, AAA activity of AChE is inhibited by the active site inhibitors while remaining unaffected by the peripheral anionic site inhibitors. This differential inhibitory pattern of active and peripheral anionic site inhibitors on the AAA activity remains unanswered. To answer this, we investigated the mechanism of binding and trafficking of AAA substrates using in silico tools. Molecular docking of serotonin and AAA substrates (o-nitroacetanilide, and o-nitrotrifluoroacetanilide,) onto AChE shows that these compounds bind at the side door of AChE. Thus, we conceived that the AAA substrates prefer the side door to reach the active site for their catalysis. Further, steered molecular dynamics simulations show that the force required for binding and trafficking of the AAA substrate through the side door is comparatively lesser than their dissociation (900kJ/mol/nm). Among the two substrates, o-nitrotrifluoroacetanilide required lesser force (380kJ/mol/nm) than o-nitroacetanilide the (550kJ/mol/nm) for its binding, thus validating o-nitrotrifluoroacetanilide as a better substrate. With these observations, we resolve that the AAA activity of AChE is mediated through its side door. Therefore, binding of PAS inhibitors at the main door of AChE remain ineffective against AAA activity.

Besides esterase activity, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) hydrolyze o-nitroacetanilides through aryl acylamidase activity. We have reported that BuChE tetramers and monomers of human blood plasma differ in o-nitroacetanilide (ONA) hydrolysis. The homology in quaternary structure and folding of subunits in the prevalent BuChE species (G4(H)) of human plasma and AChE forms of fetal bovine serum prompted us to study the esterase and amidase activities of fetal bovine serum AChE. The k(cat)/K(m) values for acetylthiocholine (ATCh), ONA and its trifluoro derivative N-(2-nitrophenyl)-trifluoroacetamide (F-ONA) were 398 x 10(6) M(-1) min(-1), 0.8 x 10(6) M(-1) min(-1), and 17.5 x 10(6) M(-1) min(-1), respectively. The lack of inhibition of amidase activity at high F-ONA concentrations makes it unlikely that there is a role for the peripheral anionic site (PAS) in F-ONA degradation, but the inhibition of ATCh, ONA and F-ONA hydrolysis by the PAS ligand fasciculin-2 points to the transit of o-nitroacetalinides near the PAS on their way to the active site. Sedimentation analysis confirmed substrate hydrolysis by tetrameric 10.9S AChE. As compared with esterase activity, amidase activity was less sensitive to guanidine hydrochloride. This reagent led to the formation of 9.3S tetramers with partially unfolded subunits. Their capacity to hydrolyze ATCh and F-ONA revealed that, despite the conformational change, the active site architecture and functionality of AChE were partially retained.

Albumin is generally regarded as an inert protein with no enzyme activity. However, albumin has esterase activity as well as aryl acylamidase activity. A new acetanilide substrate, o-nitrotrifluoroacetanilide (o-NTFNAC), which is more reactive than the classical o-nitroacetanilide, made it possible to determine the catalytic parameters for hydrolysis by fatty-acid free human serum albumin. Owing to the low enzymatic activity of albumin, kinetic studies were performed at high albumin concentration (0.075 mM). The albumin behavior with this substrate was Michaelis-Menten like. Kinetic analysis was performed according to the formalism used for catalysis at high enzyme concentration. This approach provided values for the turnover and dissociation constant of the albumin-substrate complex: k(cat) = 0.13 +/- 0.02 min(-1) and Ks = 0.67 +/- 0.04 mM. MALDI-TOF experiments showed that unlike the ester substrate p-nitrophenyl acetate, o-NTFNAC does not form a stable adduct (acetylated enzyme). Kinetic analysis and MALDI-TOF experiments demonstrated that hydrolysis of o-NTFNAC by albumin is fully rate-limited by the acylation step (k(cat) = k2). Though the aryl acylamidase activity of albumin is low (k(cat)/Ks = 195 M(-1)min(-1)), because of its high concentration in human plasma (0.6-1 mM), albumin may participate in hydrolysis of aryl acylamides through second-order kinetics. This suggests that albumin may have a role in the metabolism of endogenous and exogenous aromatic amides, including drugs and xenobiotics.

Acetylcholinesterase (AChE) exhibits two different activities, namely esterase and aryl acylamidase (AAA). Unlike esterase, AAA activity of AChE is inhibited by the active site inhibitors while remaining unaffected by the peripheral anionic site inhibitors. This differential inhibitory pattern of active and peripheral anionic site inhibitors on the AAA activity remains unanswered. To answer this, we investigated the mechanism of binding and trafficking of AAA substrates using in silico tools. Molecular docking of serotonin and AAA substrates (o-nitroacetanilide, and o-nitrotrifluoroacetanilide,) onto AChE shows that these compounds bind at the side door of AChE. Thus, we conceived that the AAA substrates prefer the side door to reach the active site for their catalysis. Further, steered molecular dynamics simulations show that the force required for binding and trafficking of the AAA substrate through the side door is comparatively lesser than their dissociation (900kJ/mol/nm). Among the two substrates, o-nitrotrifluoroacetanilide required lesser force (380kJ/mol/nm) than o-nitroacetanilide the (550kJ/mol/nm) for its binding, thus validating o-nitrotrifluoroacetanilide as a better substrate. With these observations, we resolve that the AAA activity of AChE is mediated through its side door. Therefore, binding of PAS inhibitors at the main door of AChE remain ineffective against AAA activity.

Besides esterase activity, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) hydrolyze o-nitroacetanilides through aryl acylamidase activity. We have reported that BuChE tetramers and monomers of human blood plasma differ in o-nitroacetanilide (ONA) hydrolysis. The homology in quaternary structure and folding of subunits in the prevalent BuChE species (G4(H)) of human plasma and AChE forms of fetal bovine serum prompted us to study the esterase and amidase activities of fetal bovine serum AChE. The k(cat)/K(m) values for acetylthiocholine (ATCh), ONA and its trifluoro derivative N-(2-nitrophenyl)-trifluoroacetamide (F-ONA) were 398 x 10(6) M(-1) min(-1), 0.8 x 10(6) M(-1) min(-1), and 17.5 x 10(6) M(-1) min(-1), respectively. The lack of inhibition of amidase activity at high F-ONA concentrations makes it unlikely that there is a role for the peripheral anionic site (PAS) in F-ONA degradation, but the inhibition of ATCh, ONA and F-ONA hydrolysis by the PAS ligand fasciculin-2 points to the transit of o-nitroacetalinides near the PAS on their way to the active site. Sedimentation analysis confirmed substrate hydrolysis by tetrameric 10.9S AChE. As compared with esterase activity, amidase activity was less sensitive to guanidine hydrochloride. This reagent led to the formation of 9.3S tetramers with partially unfolded subunits. Their capacity to hydrolyze ATCh and F-ONA revealed that, despite the conformational change, the active site architecture and functionality of AChE were partially retained.

Albumin is generally regarded as an inert protein with no enzyme activity. However, albumin has esterase activity as well as aryl acylamidase activity. A new acetanilide substrate, o-nitrotrifluoroacetanilide (o-NTFNAC), which is more reactive than the classical o-nitroacetanilide, made it possible to determine the catalytic parameters for hydrolysis by fatty-acid free human serum albumin. Owing to the low enzymatic activity of albumin, kinetic studies were performed at high albumin concentration (0.075 mM). The albumin behavior with this substrate was Michaelis-Menten like. Kinetic analysis was performed according to the formalism used for catalysis at high enzyme concentration. This approach provided values for the turnover and dissociation constant of the albumin-substrate complex: k(cat) = 0.13 +/- 0.02 min(-1) and Ks = 0.67 +/- 0.04 mM. MALDI-TOF experiments showed that unlike the ester substrate p-nitrophenyl acetate, o-NTFNAC does not form a stable adduct (acetylated enzyme). Kinetic analysis and MALDI-TOF experiments demonstrated that hydrolysis of o-NTFNAC by albumin is fully rate-limited by the acylation step (k(cat) = k2). Though the aryl acylamidase activity of albumin is low (k(cat)/Ks = 195 M(-1)min(-1)), because of its high concentration in human plasma (0.6-1 mM), albumin may participate in hydrolysis of aryl acylamides through second-order kinetics. This suggests that albumin may have a role in the metabolism of endogenous and exogenous aromatic amides, including drugs and xenobiotics.

The aryl-acylamidase (AAA) activity of butyrylcholinesterase (BuChE) has been known for a long time. However, the kinetic mechanism of aryl-acylamide hydrolysis by BuChE has not been investigated. Therefore, the catalytic properties of human BuChE and its peripheral site mutant (D70G) toward neutral and charged aryl-acylamides were determined. Three neutral (o-nitroacetanilide, m-nitroacetanilide, o-nitrophenyltrifluoroacetamide) and one positively charged (3-(acetamido) N,N,N-trimethylanilinium, ATMA) acetanilides were studied. Hydrolysis of ATMA by wild-type and D70G enzymes showed a long transient phase preceding the steady state. The induction phase was characterized by a hysteretic "burst". This reflects the existence of two enzyme states in slow equilibrium with different catalytic properties. Steady-state parameters for hydrolysis of the three acetanilides were compared to catalytic parameters for hydrolysis of esters giving the same acetyl intermediate. Wild-type BuChE showed substrate activation while D70G displayed a Michaelian behavior with ATMA as with positively charged esters. Owing to the low affinity of BuChE for amide substrates, the hydrolysis kinetics of neutral amides was first order. Acylation was the rate-determining step for hydrolysis of aryl-acetylamide substrates. Slow acylation of the enzyme, relative to that by esters may, in part, be due suboptimal fit of the aryl-acylamides in the active center of BuChE. The hypothesis that AAA and esterase active sites of BuChE are non-identical was tested with mutant BuChE. It was found that mutations on the catalytic serine, S198C and S198D, led to complete loss of both activities. The silent variant (FS117) had neither esterase nor AAA activity. Mutation in the peripheral site (D70G) had the same effect on esterase and AAA activities. Echothiophate inhibited both activities identically. It was concluded that the active sites for esterase and AAA activities are identical, i.e. S198. This excludes any other residue present in the gorge for being the catalytic nucleophile pole.

Cholinesterases, in addition to their well-known esterase action, also show an aryl acylamidase (AAA) activity whereby they catalyze the hydrolysis of amides of certain aromatic amines. The biological function of this catalysis is not known. Furthermore, it is not known whether the esterase catalytic site is involved in the AAA activity of cholinesterases. It has been speculated that the AAA activity, especially that of butyrylcholinesterase (BuChE), may be important in the development of the nervous system and in pathological processes such as formation of neuritic plaques in Alzheimer's disease (AD). The substrate generally used to study the AAA activity of cholinesterases is N-(2-nitrophenyl)acetamide. However, use of this substrate requires high concentrations of enzyme and substrate, and prolonged periods of incubation at elevated temperature. As a consequence, difficulties in performing kinetic analysis of AAA activity associated with cholinesterases have hampered understanding this activity. Because of its potential biological importance, we sought to develop a more efficient and specific substrate for use in studying the AAA activity associated with BuChE, and for exploring the catalytic site for this hydrolysis. Here, we describe the structure-activity relationships for hydrolysis of anilides by cholinesterases. These studies led to a substrate, N-(2-nitrophenyl)trifluoroacetamide, that was hydrolyzed several orders of magnitude faster than N-(2-nitrophenyl)acetamide by cholinesterases. Also, larger N-(2-nitrophenyl)alkylamides were found to be more rapidly hydrolyzed by BuChE than N-(2-nitrophenyl)acetamide and, in addition, were more specific for hydrolysis by BuChE. Thus, N-(2-nitrophenyl)alkylamides with six to eight carbon atoms in the acyl group represent suitable specific substrates to investigate further the function of the AAA activity of BuChE. Based on the substrate structure-activity relationships and kinetic studies, the hydrolysis of anilides and esters of choline appears to utilize the same catalytic site in BuChE.