Conversion of acetylcholinesterase to butyrylcholinesterase: modeling and mutagenesis.

Abstract

Torpedo acetyicholinesterase (AcChoEase, EC 3.1.1.7) and human butyrylcholnesterase (BtChoEase, EC 3.1.1.8), while clearly differing in substrate specificity and sensitivity to inhibitors, possess 53% sequence homology; this permitted modeling human BtChoEase on the basis of the three-dimensional structure of Torpedo AcChoEase. The modeled BtChoEase structure closely resembled that of AcChoEase in overall features. However, six conserved aromatic residues that line the active-site gorge, which is a prominent feature ofthe AcChoEase structure, are absent in BtChoEase. Modeling showed that two such residues, Phe-288 and Phe-290, replaced by leucine and valine, respectively, in BtChoEase, may prevent entrance of butyrylcholine into the acyl-binding pocket. Their mutation to leucine and valine in AcChoEase, by site-directed mutagenesis, produced a double mutant that hydrolyzed butyrylthiocholine almost as well as acetylthiocholine. The mutated enzyme was also inhibited well by the bulky, BtChoEaseselective organophosphate inhibitor (tetraisopropylpyrophosphoramide, iso-OMPA). Trp-279, at the entrance of the activesite gorge in AcChoEase, is absent in BtChoEase. Modeling designated it as part of the "peripheral" anionic site, which is lacking in BtChoEase. The mutant W279A displayed strongly reduced inhibition by the peripheral site-specific ligand propidium relative to wild-type Torpedo AcChoEase, whereas inhibition by the catalytic-site inhibitor edrophonium was unaffected.

Cholinesterases, a Target of Pharmacology and Toxicology

“…Substitution of two phenylalanines to leucine and valine in the electric eel AChE aromatic gorge possessed butyrylcholine turnover rate at a similar level to BuChE. Further, mutated AChE was sensitive to inhibition by iso-OMPA and was not inhibited by propidium, a peripheral anionic site inhibitor 61 . Differences in AChE and BuChE structures are revealed by huperzine A.…”

Structure of a complex of the potent and specific inhibitor BW284C51 withTorpedo californicaacetylcholinesterase

“…The three-dimensional structure of Torpedo californica AChE (TcAChE) revealed that its active site is located at the bottom of a long and narrow cavity lined with aromatic residues, which was named the`active-site gorge' (Sussman et al, 1991). The active site contains a catalytic triad and an oxyanion hole, similar to those found in other serine hydrolases (Steitz & Shulman, 1982), an acyl pocket which recognizes the acetyl group (Harel et al, 1992) and a so-called`anionic site', which recognizes the quaternary group of ACh. Surprisingly, the principal component of this anionic site is not a cluster of negative charges, as previously postulated (Nolte et al, 1980), but rather the indole moiety of Trp84, which makes a %±cation interaction (Dougherty & Stauffer, 1990;Felder et al, 2001) with the quaternary group of ACh (Sussman et al, 1991).…”

“…This led to the de®nition of a socalled`peripheral' anionic binding site, with the assumption that the bisquaternary ligands span the two anionic sites. The X-ray structure of the complex of the bisquaternary ligand decamethonium with TcAChE, together with site-directed mutagenesis (Harel et al, 1992(Harel et al, , 1993, clearly showed this peripheral site to be located close to the mouth of the activesite gorge. Furthermore, aromatic residues, principally Tyr70 and Trp279, were again shown to be important elements of this so-called`anionic' site.…”

Recovery of Acetylcholinesterase Activity in the Common Carp ( Cyprinus carpio L.) After Inhibition by Organophosphate and Carbamate Compounds

“…There are two distinct types of enzymes capable of acetylcholine hydrolysis. The acetylcholinesterase or "true cholinesterase" (AChE EC.3.1.1.8) predominantly associated with nervous tissue and erythrocytes, and the pseudo cholinesterase (ChE EC: 3.1.1.7) which is found in the serum, muscle and ganglia (Fossi et al 1995;Harel et al 1992;Vigny et al 1978). These two enzymes, AChE and ChE can be distinguished not only by their distribution but also by their substrate specificity and their sensitivity to certain selective inhibitors (Augustisson and Nachmanshon 1949;Davison 1955;Toutant et al 1985;Vigny et al 1978).…”