Abstract
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are thought to be the result of a gene duplication event early in vertebrate evolution. To learn more about the evolution of these enzymes, we expressed in vitro, characterized, and modeled a recombinant cholinesterase (ChE) from a teleost, the medaka Oryzias latipes. In addition to AChE, O. latipes has a ChE that is different from either vertebrate AChE or BChE, which we are classifying as an atypical BChE, and which may resemble a transitional form between the two. Of the fourteen aromatic amino acids in the catalytic gorge of vertebrate AChE, ten are conserved in the atypical BChE of O. latipes; by contrast, only eight are conserved in vertebrate BChE. Notably, the atypical BChE has one phenylalanine in its acyl pocket, while AChE has two and BChE none. These substitutions could account for the intermediate nature of this atypical BChE. Molecular modeling supports this proposal. The atypical BChE hydrolyzes acetylthiocholine (ATCh) and propionylthiocholine (PTCh) preferentially but butyrylthiocholine (BTCh) to a considerable extent, which is different from the substrate specificity of AChE or BChE. The enzyme shows substrate inhibition with the two smaller substrates but not with the larger substrate BTCh. In comparison, AChE exhibits substrate inhibition, while BChE does not, but may instead show substrate activation. The atypical BChE from O. latipes also shows a mixed pattern of inhibition. It is effectively inhibited by physostigmine, typical of all ChEs. However, although the atypical BChE is efficiently inhibited by the BChE-specific inhibitor ethopropazine, it is not by another BChE inhibitor, iso-OMPA, nor by the AChE-specific inhibitor BW284c51. The atypical BChE is found as a glycophosphatidylinositol-anchored (GPI-anchored) amphiphilic dimer (G2 a), which is unusual for any BChE. We classify the enzyme as an atypical BChE and discuss its implications for the evolution of AChE and BChE and for ecotoxicology.
Highlights
Acetylcholinesterase (AChE; EC 3.1.1.7) hydrolyzes acetylcholine at the neuromuscular junction of vertebrates
Sequence Analysis Reveals Two ChEs in O. latipes Two expressed sequences for ChEs are present in the O. latipes genome: AChE (GenBank EST DK110600) and an enzyme that we are classifying as an atypical BChE [45] (GenBank cDNAs AV668390 and GU797251)
Pair-wise BLAST alignments of sequences from the catalytic region of ChEs show that the AChE from O. latipes clearly resembles T. californica AChE rather than Homo sapiens BChE (68/80% identity/similarity to AChE compared to 54/70% for BChE), while the atypical
Summary
Acetylcholinesterase (AChE; EC 3.1.1.7) hydrolyzes acetylcholine at the neuromuscular junction of vertebrates. Higher vertebrates contain an evolutionarily related cholinesterase (ChE), butyrylcholinesterase (BChE, EC 3.1.1.8). The function of BChE is unknown but is suggested to play a role in growth and development and to act as a scavenger of cholinergic toxins as well as having an auxiliary role in synaptic transmission [1,2]. The two ChEs may be distinguished functionally both kinetically and pharmacologically: AChE hydrolyzes acetylcholine (ACh) and is virtually inactive on the larger substrate butyrylcholine (BCh). BChE is less selective, hydrolyzing both substrates comparably. AChE exhibits inhibition at high substrate concentrations, while BChE shows substrate activation instead [3]. The two enzymes may be distinguished by their susceptibility to diagnostic inhibitors [4]
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