Chemical modification of acetylcholinesterase from eel and basal ganglia: effect on the acetylcholinesterase and aryl acylamidase activities

Abstract

The effect of chemical modification on the acetylcholinesterase and the aryl acylamidase activities of purified acetylcholinesterase from electric eel and basal ganglia was investigated in the presence and absence of acetylcholine, the substrate of acetylcholinesterase, and 1,5-bis[4-(allyldimethylammonium)phenyl]pentan-3-one dibromide (BW284C51), a reversible competitive inhibitor of acetylcholinesterase. Trinitrobenzenesulfonic acid, pyridoxal phosphate, acetic anhydride, diethyl pyrocarbonate, and 2-hydroxy-5-nitrobenzyl bromide under specified conditions inactivated both acetylcholinesterase and aryl acylamidase in the absence of acetylcholine and BW284C51. Chemical modifications in the presence of acetylcholine and BW284C51 by all the above except diethyl pyrocarbonate selectively prevented the loss of acetylcholinesterase but not aryl acylamidase activity; modification by diethyl pyrocarbonate in the presence of acetylcholine and BW284C51 prevented the loss of both acetylcholinesterase and aryl acylamidase activities. Treatment with N-acetylimidazole resulted in the inactivation of acetylcholinesterase and the activation of aryl acylamidase. These changes in both the activities could be prevented by acetylcholine and BW284C51. Modification by phenylglyoxal, 2,4-pentanedione, or N-ethylmaleimide did not affect the enzyme activities. Indophenylacetate hydrolase activity followed a pattern similar to that of acetylcholinesterase in all the above modification studies. The results suggested essential lysine, tyrosine, tryptophan, and histidine residues for the active center of acetylcholinesterase and essential lysine, histidine, and tryptophan residues for the active center of aryl acylamidase.(ABSTRACT TRUNCATED AT 250 WORDS)

Intramolecular relationships in cholinesterases revealed by oocyte expression of site-directed and natural variants of human BCHE.

“…The influence of such hydrophobic interactions in BCHE may explain the minimal 10-fold increase in BenzCh binding to all BCHE enzymes in comparison with other choline esters. Furthermore, the selective labelling of the cationic, irreversible alkylating agent N,N-dimethyl-2-phenylaziridinum (DPA) to Trp84 in Torpedo ACHE (Weise et al, 1990), also indicates the likely role of aromatic residues in constituting the CHE binding domain, supporting previous biophysical (Shinitzky et al, 1973;Berman et al, 1985) and biochemical (Majumdar and Balasubramanian, 1984;Page and Wilson, 1985) studies. In addition, X-ray crystallography data for human lipase, also functioning by a catalytic triad, reveals Trp and Tyr residues in close apposition to the nucleophilic active centre serine (Brady et al, 1990;Winkler et al, 1990).…”

Monoclonal anti-idiotypic antibodies as functional internal images of enzyme active sites: production of a catalytic antibody with a cholinesterase activity.

“…Inactivation of AcChoE by chemical modification of histidine residues with diethyl pyrocarbonate has been described (11). Incubation of mAb 9A8 with 2.5 mM diethyl pyrocarbonate for 20 min resulted in the total loss ofcatalytic activity.…”

“…Chemical modification of histidine residues by diethyl pyrocarbonate was performed by following a procedure described for AcChoE (11). mAb (175 ,ug) and diethyl pyrocarbonate (2.5 mM) in 500 A of 10 mM phosphate buffer (pH 7.4) were incubated at 4°C for 20 min.…”

Mutagenesis of essential functional residues in acetylcholinesterase.

“…Indirect evidence, including a pH dependence for catalysis of neutral and charged substrates (33), inhibition by ethoxyformic anhydride and diethyl pyrocarbonate (34,35), residual reaction with diisopropyl fluorophosphate (8), and retention of a proton donor-acceptor function for the nucleophile in a variety of serine and cysteine hydrolases, indicates a role for a proximal histidine in catalysis. Sequence analysis of cholinesterases from Drosophila to humans delineates only two histidines conserved at positions corresponding to residues 425 and 440 in Torpedo (refs.…”