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Evolution of acetylcholinesterase and butyrylcholinesterase in the vertebrates: an atypical butyrylcholinesterase from the Medaka Oryzias latipes.

Pezzementi L, Nachon F, Chatonnet A - PLoS ONE (2011)

Bottom Line: 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 (G(2) (a)), which is unusual for any BChE.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Birmingham-Southern College, Birmingham, Alabama, United States of America. lpezzeme@bsc.edu

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 (G(2) (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.

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Substrate concentration dependencies for hydrolysis of ATCh, PTCh, and BTCh by recombinant BChE from O. latipes.Transfected COS-7 cells producing BChE were extracted in HIS buffer and assayed with ATCh (•), PTCh (□), or BTCh (▴) and fit as described in Materials and Methods.
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pone-0017396-g005: Substrate concentration dependencies for hydrolysis of ATCh, PTCh, and BTCh by recombinant BChE from O. latipes.Transfected COS-7 cells producing BChE were extracted in HIS buffer and assayed with ATCh (•), PTCh (□), or BTCh (▴) and fit as described in Materials and Methods.

Mentions: As we were interested in the functional characteristics of the atypical BChE in O. latipes, we cloned and expressed in vitro a cDNA for the enzyme in COS-7 cells. To determine the substrate specificity of the enzyme, we assayed the hydrolysis of the substrates acetylthiocholine (ATCh), propionylthiocholine (PTCh), and butyrylthiocholine (BTCh) by the recombinant enzyme (Fig. 5). The smaller substrates ATCh and PTCh are hydrolyzed more or less equally, as indicated by the similar values of kcatSubstrate/kcatATCh (Table 3); the larger BTCh is hydrolyzed at about a quarter of the rate of the other two substrates. The Kms are inversely proportional to the length of the acyl group, with BTCh having the lowest Km. The highest catalytic efficiency (kcat/Km) is seen with PTCh. Additionally, ATCh and PTCh produce substrate inhibition (i.e., lower enzyme activity at high substrate concentrations and b parameter values of <1), although the inhibition by PTCh is weak (Fig. 5; Table 3). BTCh does not produce substrate inhibition. Overall, this pattern of substrate hydrolysis is not typical of either AChE or BChE.


Evolution of acetylcholinesterase and butyrylcholinesterase in the vertebrates: an atypical butyrylcholinesterase from the Medaka Oryzias latipes.

Pezzementi L, Nachon F, Chatonnet A - PLoS ONE (2011)

Substrate concentration dependencies for hydrolysis of ATCh, PTCh, and BTCh by recombinant BChE from O. latipes.Transfected COS-7 cells producing BChE were extracted in HIS buffer and assayed with ATCh (•), PTCh (□), or BTCh (▴) and fit as described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045457&req=5

pone-0017396-g005: Substrate concentration dependencies for hydrolysis of ATCh, PTCh, and BTCh by recombinant BChE from O. latipes.Transfected COS-7 cells producing BChE were extracted in HIS buffer and assayed with ATCh (•), PTCh (□), or BTCh (▴) and fit as described in Materials and Methods.
Mentions: As we were interested in the functional characteristics of the atypical BChE in O. latipes, we cloned and expressed in vitro a cDNA for the enzyme in COS-7 cells. To determine the substrate specificity of the enzyme, we assayed the hydrolysis of the substrates acetylthiocholine (ATCh), propionylthiocholine (PTCh), and butyrylthiocholine (BTCh) by the recombinant enzyme (Fig. 5). The smaller substrates ATCh and PTCh are hydrolyzed more or less equally, as indicated by the similar values of kcatSubstrate/kcatATCh (Table 3); the larger BTCh is hydrolyzed at about a quarter of the rate of the other two substrates. The Kms are inversely proportional to the length of the acyl group, with BTCh having the lowest Km. The highest catalytic efficiency (kcat/Km) is seen with PTCh. Additionally, ATCh and PTCh produce substrate inhibition (i.e., lower enzyme activity at high substrate concentrations and b parameter values of <1), although the inhibition by PTCh is weak (Fig. 5; Table 3). BTCh does not produce substrate inhibition. Overall, this pattern of substrate hydrolysis is not typical of either AChE or BChE.

Bottom Line: 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 (G(2) (a)), which is unusual for any BChE.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Birmingham-Southern College, Birmingham, Alabama, United States of America. lpezzeme@bsc.edu

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 (G(2) (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.

Show MeSH
Related in: MedlinePlus