Characterization of a high-activity mutant of human butyrylcholinesterase against (-)-cocaine

Wenchao Yang; Liu Xue; Lei Fang; Xi Chen; Chang Guo Zhan

doi:10.1016/j.cbi.2010.01.004

Characterization of a high-activity mutant of human butyrylcholinesterase against (-)-cocaine

Wenchao Yang, Liu Xue, Lei Fang, Xi Chen, Chang Guo Zhan

Pharmaceutical Sciences

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61 Scopus citations

Abstract

Cocaine addiction and overdose are a well-known public health problem. There is no approved medication available for cocaine abuse treatment. Our recently designed and discovered high-activity mutant (A199S/S287G/A328W/Y332G) of human butyrylcholinesterase (BChE) has been recognized to be worth exploring for clinical application in humans as a potential anti-cocaine medication. The catalytic rate constant (k_cat) and Michaelis-Menten constant (K_M) for (-)-cocaine hydrolysis catalyzed by A199S/S287G/A328W/Y332G BChE (without fusion with any other peptide) have been determined to be 3060min^-1 and 3.1μM, respectively, in the present study. The determined kinetic parameters reveal that the un-fused A199S/S287G/A328W/Y332G mutant has a ∼1080-fold improved catalytic efficiency (k_cat/K_M) against (-)-cocaine compared to the wild-type BChE. The ∼1080-fold improvement in the catalytic efficiency of the un-fused A199S/S287G/A328W/Y332G mutant is very close to the previously reported the ∼1000-fold improvement in the catalytic efficiency of the A199S/S287G/A328W/Y332G mutant fused with human serum albumin. These results suggest that the albumin fusion did not significantly change the catalytic efficiency of the BChE mutant while extending the plasma half-life. In addition, we have also examined the catalytic activities of the A199S/S287G/A328W/Y332G mutant against two other substrates, acetylthiocholine (ATC) and butyrylthiocholine (BTC). It has been shown that the A199S/S287G/A328W/Y332G mutations actually decreased the catalytic efficiencies of BChE against ATC and BTC, while considerably improving the catalytic efficiency of BChE against (-)-cocaine.

Original language	English
Pages (from-to)	148-152
Number of pages	5
Journal	Chemico-Biological Interactions
Volume	187
Issue number	1-3
DOIs	https://doi.org/10.1016/j.cbi.2010.01.004
State	Published - Sep 2010

Bibliographical note

Funding Information:
This work was supported by NIH (grants R01 DA021416 , R01 DA025100 , and R01 DA013930 ). The authors also acknowledge the Center for Computational Sciences (CCS) at University of Kentucky for supercomputing time on an IBM X-series Cluster with 1360 processors.

Funding

This work was supported by NIH (grants R01 DA021416 , R01 DA025100 , and R01 DA013930 ). The authors also acknowledge the Center for Computational Sciences (CCS) at University of Kentucky for supercomputing time on an IBM X-series Cluster with 1360 processors.

Funders	Funder number
National Institutes of Health (NIH)	R01 DA013930, R01 DA025100
National Institute on Drug Abuse	R01DA021416

Keywords

Cocaine addiction
Drug overdose
Enzyme therapy
Hydrolase

ASJC Scopus subject areas

Toxicology

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.cbi.2010.01.004

Cite this

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title = "Characterization of a high-activity mutant of human butyrylcholinesterase against (-)-cocaine",

abstract = "Cocaine addiction and overdose are a well-known public health problem. There is no approved medication available for cocaine abuse treatment. Our recently designed and discovered high-activity mutant (A199S/S287G/A328W/Y332G) of human butyrylcholinesterase (BChE) has been recognized to be worth exploring for clinical application in humans as a potential anti-cocaine medication. The catalytic rate constant (kcat) and Michaelis-Menten constant (KM) for (-)-cocaine hydrolysis catalyzed by A199S/S287G/A328W/Y332G BChE (without fusion with any other peptide) have been determined to be 3060min-1 and 3.1μM, respectively, in the present study. The determined kinetic parameters reveal that the un-fused A199S/S287G/A328W/Y332G mutant has a ∼1080-fold improved catalytic efficiency (kcat/KM) against (-)-cocaine compared to the wild-type BChE. The ∼1080-fold improvement in the catalytic efficiency of the un-fused A199S/S287G/A328W/Y332G mutant is very close to the previously reported the ∼1000-fold improvement in the catalytic efficiency of the A199S/S287G/A328W/Y332G mutant fused with human serum albumin. These results suggest that the albumin fusion did not significantly change the catalytic efficiency of the BChE mutant while extending the plasma half-life. In addition, we have also examined the catalytic activities of the A199S/S287G/A328W/Y332G mutant against two other substrates, acetylthiocholine (ATC) and butyrylthiocholine (BTC). It has been shown that the A199S/S287G/A328W/Y332G mutations actually decreased the catalytic efficiencies of BChE against ATC and BTC, while considerably improving the catalytic efficiency of BChE against (-)-cocaine.",

keywords = "Cocaine addiction, Drug overdose, Enzyme therapy, Hydrolase",

author = "Wenchao Yang and Liu Xue and Lei Fang and Xi Chen and Zhan, {Chang Guo}",

note = "Funding Information: This work was supported by NIH (grants R01 DA021416 , R01 DA025100 , and R01 DA013930 ). The authors also acknowledge the Center for Computational Sciences (CCS) at University of Kentucky for supercomputing time on an IBM X-series Cluster with 1360 processors.",

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AU - Yang, Wenchao

AU - Xue, Liu

AU - Fang, Lei

AU - Chen, Xi

AU - Zhan, Chang Guo

N1 - Funding Information: This work was supported by NIH (grants R01 DA021416 , R01 DA025100 , and R01 DA013930 ). The authors also acknowledge the Center for Computational Sciences (CCS) at University of Kentucky for supercomputing time on an IBM X-series Cluster with 1360 processors.

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N2 - Cocaine addiction and overdose are a well-known public health problem. There is no approved medication available for cocaine abuse treatment. Our recently designed and discovered high-activity mutant (A199S/S287G/A328W/Y332G) of human butyrylcholinesterase (BChE) has been recognized to be worth exploring for clinical application in humans as a potential anti-cocaine medication. The catalytic rate constant (kcat) and Michaelis-Menten constant (KM) for (-)-cocaine hydrolysis catalyzed by A199S/S287G/A328W/Y332G BChE (without fusion with any other peptide) have been determined to be 3060min-1 and 3.1μM, respectively, in the present study. The determined kinetic parameters reveal that the un-fused A199S/S287G/A328W/Y332G mutant has a ∼1080-fold improved catalytic efficiency (kcat/KM) against (-)-cocaine compared to the wild-type BChE. The ∼1080-fold improvement in the catalytic efficiency of the un-fused A199S/S287G/A328W/Y332G mutant is very close to the previously reported the ∼1000-fold improvement in the catalytic efficiency of the A199S/S287G/A328W/Y332G mutant fused with human serum albumin. These results suggest that the albumin fusion did not significantly change the catalytic efficiency of the BChE mutant while extending the plasma half-life. In addition, we have also examined the catalytic activities of the A199S/S287G/A328W/Y332G mutant against two other substrates, acetylthiocholine (ATC) and butyrylthiocholine (BTC). It has been shown that the A199S/S287G/A328W/Y332G mutations actually decreased the catalytic efficiencies of BChE against ATC and BTC, while considerably improving the catalytic efficiency of BChE against (-)-cocaine.

AB - Cocaine addiction and overdose are a well-known public health problem. There is no approved medication available for cocaine abuse treatment. Our recently designed and discovered high-activity mutant (A199S/S287G/A328W/Y332G) of human butyrylcholinesterase (BChE) has been recognized to be worth exploring for clinical application in humans as a potential anti-cocaine medication. The catalytic rate constant (kcat) and Michaelis-Menten constant (KM) for (-)-cocaine hydrolysis catalyzed by A199S/S287G/A328W/Y332G BChE (without fusion with any other peptide) have been determined to be 3060min-1 and 3.1μM, respectively, in the present study. The determined kinetic parameters reveal that the un-fused A199S/S287G/A328W/Y332G mutant has a ∼1080-fold improved catalytic efficiency (kcat/KM) against (-)-cocaine compared to the wild-type BChE. The ∼1080-fold improvement in the catalytic efficiency of the un-fused A199S/S287G/A328W/Y332G mutant is very close to the previously reported the ∼1000-fold improvement in the catalytic efficiency of the A199S/S287G/A328W/Y332G mutant fused with human serum albumin. These results suggest that the albumin fusion did not significantly change the catalytic efficiency of the BChE mutant while extending the plasma half-life. In addition, we have also examined the catalytic activities of the A199S/S287G/A328W/Y332G mutant against two other substrates, acetylthiocholine (ATC) and butyrylthiocholine (BTC). It has been shown that the A199S/S287G/A328W/Y332G mutations actually decreased the catalytic efficiencies of BChE against ATC and BTC, while considerably improving the catalytic efficiency of BChE against (-)-cocaine.

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Characterization of a high-activity mutant of human butyrylcholinesterase against (-)-cocaine

Abstract

Bibliographical note

Funding

Keywords

ASJC Scopus subject areas

UN SDGs

Access to Document

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