Soluble monomeric acetylcholinesterase from mouse: expression, purification, and crystallization in complex with fasciculin.

A soluble, monomeric form of acetylcholinesterase from mouse (mAChE), truncated at its carboxyl-terminal end, was generated from a cDNA encoding the glycophospholipid-linked form of the mouse enzyme by insertion of an early stop codon at position 549. Insertion of the cDNA behind a cytomegalovirus promoter and selection by aminoglycoside resistance in transfected HEK cells yielded clones secreting large quantities of mAChE into the medium. The enzyme sediments as a soluble monomer at 4.

Allosteric control of acetylcholinesterase catalysis by fasciculin.

The interaction of fasciculin 2 was examined with wild-type and several mutant forms of acetylcholinesterase (AChE) where Trp86, which lies at the base of the active center gorge, is replaced by Tyr, Phe, and Ala. The fasciculin family of peptides from snake venom bind to a peripheral site near the rim of the gorge, but at a position which still allows substrates and other inhibitors to enter the gorge. The interaction of a series of charged and uncharged carboxyl esters, alkyl phosphoryl esters, and substituted trifluoroacetophenones were analyzed with the wild-type and mutant AChEs in the presence and absence of fasciculin.

Amino acid residues controlling reactivation of organophosphonyl conjugates of acetylcholinesterase by mono- and bisquaternary oximes.

Single and multiple site mutants of recombinant mouse acetylcholinesterase (rMoAChE) were inhibited with racemic 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide (MEPQ) and the resulting mixture of two enantiomers, CH3PR,S(O)(OC2H5)-AChE(EMPR,S-AChE), were subjected to reactivation with 2-(hydroxyiminomethyl)-1-methylpyridinium methanesulfonate (P2S) and 1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4"-carbamoyl-1"- pyridinium)-2-oxapropane dichloride (HI-6). Kinetic analysis of the reactivation profiles revealed biphasic behavior with an approximate 1:1 ratio of two presumed reactivatable enantiomeric components. Equilibrium dissociation and kinetic rate constants for reactivation of site-specific mutant enzymes were compared with those obtained for wild-type rMoAChE, tissue-derived Torpedo AChE and human plasma butyrylcholinesterase.

Identification of amino acid residues involved in the binding of Huperzine A to cholinesterases.

Huperzine A, a potential agent for therapy in Alzheimer's disease and for prophylaxis of organophosphate toxicity, has recently been characterized as a reversible inhibitor of cholinesterases. To examine the specificity of this novel compound in more detail, we have examined the interaction of the 2 stereoisomers of Huperzine A with cholinesterases and site-specific mutants that detail the involvement of specific amino acid residues. Inhibition of fetal bovine serum acetylcholinesterase by (-)-Huperzine A was 35-fold more potent than (+)-Huperzine A, with KI values of 6.

Expression and ligand specificity of acetylcholinesterase and the nicotinic receptor: a tale of two cholinergic sites.

The functional design of the nAChR and AChE rather than their recognition capacities requires divergence in structure of the two binding sites. The receptor requires co-operativity to link ligand occupation to the response, rapid conformational transitions of activation, and slower transitions of desensitization. Hence, its binding sites have evolved at subunit interfaces.