Fasciculin 2 binds to the peripheral site on acetylcholinesterase and inhibits substrate hydrolysis by slowing a step involving proton transfer during enzyme acylation. (33/38)

The acetylcholinesterase active site consists of a gorge 20 A deep that is lined with aromatic residues. A serine residue near the base of the gorge defines an acylation site where an acyl enzyme intermediate is formed during the hydrolysis of ester substrates. Residues near the entrance to the gorge comprise a peripheral site where inhibitors like propidium and fasciculin 2, a snake neurotoxin, bind and interfere with catalysis. We report here the association and dissociation rate constants for fasciculin 2 interaction with the human enzyme in the presence of ligands that bind to either the peripheral site or the acylation site. These kinetic data confirmed that propidium is strictly competitive with fasciculin 2 for binding to the peripheral site. In contrast, edrophonium, N-methylacridinium, and butyrylthiocholine bound to the acylation site and formed ternary complexes with the fasciculin 2-bound enzyme in which their affinities were reduced by about an order of magnitude from their affinities in the free enzyme. Steady state analysis of the inhibition of substrate hydrolysis by fasciculin 2 revealed that the ternary complexes had residual activity. For acetylthiocholine and phenyl acetate, saturating amounts of the toxin reduced the first-order rate constant kcat to 0.5-2% and the second-order rate constant kcat/Kapp to 0.2-2% of their values with the uninhibited enzyme. To address whether fasciculin 2 inhibition primarily involved steric blockade of the active site or conformational interaction with the acylation site, deuterium oxide isotope effects on these kinetic parameters were measured. The isotope effect on kcat/Kapp increased for both substrates when fasciculin 2 was bound to the enzyme, indicating that fasciculin 2 acts predominantly by altering the conformation of the active site in the ternary complex so that steps involving proton transfer during enzyme acylation are slowed.  (+info)

Inhibitory effects of KW-5092, a novel gastroprokinetic agent, on the activity of acetylcholinesterase in guinea pig ileum. (34/38)

KW-5092 ([1-[2-[[[5-(piperidinomethyl)-2- furanyl]methyl]amino]ethyl]-2-imidazolidinylidene] propanedinitrile fumarate) is a novel gastroprokinetic agent with acetylcholinesterase (AChE) inhibitory activity and acetylcholine release facilitatory activity. The present study used guinea pig ileal homogenates to examine the inhibitory effects of KW-5092 on the activities of AChE and butyrylcholinesterase (BuChE). KW-5092 inhibited AChE and BuChE with the IC50 values of 6.8 x 10(-8) M and 2.4 x 10(-5) M, respectively. The IC50 values of neostigmine for AChE and BuChE were 3.6 x 10(-8) M and 1.9 x 10(-7) M, respectively. HSR-803 (N-[4-[2-(dimethylamino)ethoxy]benzyl]-3,4-dimethoxybenzamide hydrochloride), a gastroprokinetic agent, inhibited AChE and BuChE with the IC50 values of 8.6 x 10(-6) M and 6.0 x 10(-4) M, respectively. The AChE inhibition by KW-5092 was reversible and noncompetitive, whereas that by HSR-803 was reversible and uncompetitive. On the other hand, the AChE inhibition by neostigmine was non-competitive when the enzyme was preincubated with this inhibitor for 2 min prior to the addition of the substrate, and it was nearly competitive when the enzyme, the inhibitor and the substrate were incubated simultaneously. The present results demonstrate that KW-5092 is a selective, reversible and noncompetitive inhibitor of AChE with different characteristics from those of neostigmine and HSR-803. The AChE inhibitory action may contribute to its gastroprokinetic effect.  (+info)

Multiple binding sites involved in the effect of choline esters on decarbamoylation of monomethylcarbamoyl- or dimethylcarbamoly-acetylcholinesterase. (35/38)

Multiple binding sites for inhibitory choline esters in spontaneous decarbamoylation of dimethylcarbamoyl-acetylcholinesterase (AChE) were suggested from a wide range of IC50 values, in contrast with a limited range of AC50 values (concentration giving 50% of maximal activation) at a peripheral activatory site. Association of choline esters containing a long acyl chain (C7-C12) with the hydrophobic zone in the active site could be deduced from a linear relationship between the size of the acyl group and the inhibitory potency in either spontaneous decarbamoylation or acetylthiocholine hydrolysis. Direct support for laurylcholine binding to the active site might come from the competitive inhibition (Ki 33 microM) of choline-catalysed decarbamoylation by laurylcholine. Moreover, its inhibitory action was greater for monomethylcarbamoyl-AChE than for dimethylcarbamoyl-AChE, where there is a greater steric hindrance at the active centre. In further support, the inhibition of pentanoylthiocholine-induced decarbamoylation by laurylcholine was suggested to be due to laurylcholine binding to a central site rather than a peripheral site, similar to the inhibition of spontaneous decarbamoylation by laurylcholine. Supportive data for acetylcholine binding to the active site are provided by the results that acetylcholine is a competitive inhibitor (Ki 7.6 mM) of choline-catalysed decarbamoylation, and its inhibitory action was greater for monomethylcarbamoyl-AChE than for dimethylcarbamoyl-AChE. Meanwhile, choline esters with an acyl group of an intermediate size (C4-C6), more subject to steric exclusion at the active centre, and less associable with the hydrophobic zone, appear to bind preferentially to a peripheral activity site. Thus the multiple effects of choline esters may be governed by hydrophobicity and/or a steric effect exerted by the acyl moiety at the binding sites.  (+info)

Site of fasciculin interaction with acetylcholinesterase. (36/38)

Fasciculin, a 6750-Da peptide from the venom of Dendroaspis, is known to inhibit reversibly mammalian and fish acetylcholinesterases at picomolar concentrations, but is a relatively weak inhibitor of avian, reptile, and insect acetylcholinesterases and mammalian butyryl-cholinesterases. An examination of fasciculin association with several mutant forms of recombinant DNA-derived acetylcholinesterase from mouse shows that it interacts with a cluster of residues near the rim of the gorge on the enzyme. The aromatic residues, Trp286, Tyr72, and Tyr124, have the most marked influence on fasciculin binding, whereas Asp74, a charged residue in the vicinity of the binding site that affects the binding of low molecular weight inhibitors, has little influence on fasciculin binding. The 3 aromatic residues are unique to the susceptible acetylcholinesterases and, along with Asp74, constitute part of the peripheral anionic site. Fasciculin falls in the family of three-loop toxins that include the receptor blocking alpha-toxins and cardiotoxins. From this basic structural motif, a binding site has evolved on fasciculin to be highly specific for the peripheral site on acetylcholinesterase. Acetylthiocholine affects rates of fasciculin binding at concentrations causing substrate inhibition. In the case of the mutant cholinesterases where rates of fasciculin dissociation are more rapid, steady state kinetic parameters also show acetylthiocholine-fasciculin competition to be consistent with occupation at a peripheral or substrate inhibition site rather than the active center.  (+info)

Electrostatic influence on the kinetics of ligand binding to acetylcholinesterase. Distinctions between active center ligands and fasciculin. (37/38)

To explore the role that surface and active center charges play in electrostatic attraction of ligands to the active center gorge of acetylcholinesterase (AChE), and the influence of charge on the reactive orientation of the ligand, we have studied the kinetics of association of cationic and neutral ligands with the active center and peripheral site of AChE. Electrostatic influences were reduced by sequential mutations of six surface anionic residues outside of the active center gorge (Glu-84, Glu-91, Asp-280, Asp-283, Glu-292, and Asp-372) and three residues within the active center gorge (Asp-74 at the rim and Glu-202 and Glu-450 at the base). The peripheral site ligand, fasciculin 2 (FAS2), a peptide of 6.5 kDa with a net charge of +4, shows a marked enhancement of rate of association with reduction in ionic strength, and this ionic strength dependence can be markedly reduced by progressive neutralization of surface and active center gorge anionic residues. By contrast, neutralization of surface residues only has a modest influence on the rate of cationic m-trimethylammoniotrifluoroacetophenone (TFK+) association with the active serine, whereas neutralization of residues in the active center gorge has a marked influence on the rate but with little change in the ionic strength dependence. Brownian dynamics calculations for approach of a small cationic ligand to the entrance of the gorge show the influence of individual charges to be in quantitative accord with that found for the surface residues. Anionic residues in the gorge may help to orient the ligand for reaction or to trap the ligand. Bound FAS2 on AChE not only reduces the rate of TFK+ reaction with the active center but inverts the ionic strength dependence for the cationic TFK+ association with AChE. Hence it appears that TFK+ must traverse an electrostatic barrier at the gorge entry imparted by the bound FAS2 with its net charge of +4.  (+info)

cDNA cloning, in vitro expression, and biochemical characterization of cholinesterase 1 and cholinesterase 2 from amphioxus--comparison with cholinesterase 1 and cholinesterase 2 produced in vivo. (38/38)

We have isolated cDNAs coding for the complete amino acid sequences of cholinesterase 1 (ChE1) and cholinesterase 2 (ChE2) from amphioxus. Both ChE transcripts have the characteristics of H-type catalytic subunits, which are inserted in the membrane via an ethanolamine-glycan-phosphatidylinositol anchor. The members of the catalytic triad of ChEs, the three pairs of cysteine residues involved in intrachain disulfide bonding, a cysteine near the carboxy terminal of both sequences, which could mediate interchain disulfide bonding, and 11 of the 14 aromatic amino acids that line the catalytic gorge of AChE are conserved. A remarkable difference between the two enzymes is in the region of the acyl-binding pocket, which plays an important role in determining substrate specificity in cholinesterases. ChE2 contains a sequence that resembles the acyl pocket of invertebrate ChE, while the acyl-binding site of ChE1 is novel. There are also differences between the two enzymes in the peripheral anionic site, which mediates inhibition by certain ligands. In vitro expression in COS-7 cells demonstrates that ChE2 hydrolyzes acetylthiocholine almost exclusively, while ChE1 hydrolyzes both acetylthiocholine and butyrylthiocholine. Both enzymes are inhibited comparably by BW284c51, but ChE1 is considerably more resistant to inhibition by propidium, ethopropazine, and eserine than is ChE2. Velocity sedimentation indicates that ChE1 and ChE2 are present as amphiphilic and nonamphiphilic G2 forms in vivo and in vitro. Another molecular form, which sediments at 17 S, is also present in vivo. Nondenaturing gel electrophoresis in conjunction with digestion by phosphatidylinositol-specific phospholipase C demonstrates that the vast majority of ChE1 and ChE2 is present as ethanolamine-glycan-phosphatidylinositol-anchored G2 forms in vivo. ChE1 also possesses an ethanolamine-glycan-phosphatidylinositol-anchor in vitro; however, ChE2 produced in vitro could not be detected on nondenaturing gels.  (+info)