Re-engineering And Efficacy Study Of Cocaine Esterase

Background:Cocaine is an alkaloid with a stimulating effect in central nervous system and extreme addictive nature.Its abuse is a global social and health problem.However,there is a lack of effective treatment for cocaine addiction and intoxication,and enzyme therapy using highly active cocaine-metabolizing enzymes to have cocaine quickly metabolized and eliminated have shown great potential in cocaine detoxification.About 45%of cocaine is metabolized by the liver to benzoylecgonine（BZE）,which is more toxic and has a longer half-life in the body compared with cocaine.It is cocaine’s main toxic metabolite that accounts for the long-term toxicity of cocaine.In our group’s previous study,bacterial cocaine esterase（Coc E）was identified as a BZE hydrolase,and its mutant V116K/T172R/G173Q/L196C/I301C（named BZEase1）has a 210-fold improvement in catalytic efficiency for BZE compared with wild type Coc E(WT Coc E,k_cat/K_M=5.84×10⁴min^-1M^-1;BZEase1,k_cat/K_M=1.43×10⁷min^-1M^-1).Moreover,BZE are structurally similar to the essential neurotransmitter acetylcholine（ACh）,and Coc E had been proved to have acetylcholinesterase activity.It is necessary to consider its potential cholinergic side effects in further development.Therefore,development of BZE-metabolizing enzyme for cocaine complete detoxification should not only have the high activity against BZE but also should have the desired substrate selectivity for BZE over ACh.Objective:This study is intended to re-engineering Coc E to further improve the catalytic activity against BZE,decrease the catalytic activity against ACh,and thus enhance the substrate selectivity through the iterative optimization strategy based on protein structure elucidation-catalytic mechanism study-rational design.Methods and results:1.The reaction mechanism of Coc E-catalyzed hydrolysis of BZE was investigated by combining the crystal structure of WT Coc E and molecular dynamics simulations,and the acylation process was determined as the reaction rate limiting step.After analysis of the interactions of key residues with substrate in the active center at the transition state,CHARMM was used to construct the initial coordinates of the mutants and the total hydrogen bonding energy of the near attack conformers of the mutants was calculated.Product ecgonine-surrounding Ala51 mutated to hydrophobic amino acid using BZEase1 which contains key mutation of V116K as the template was predicted to improve the stability of Coc E-BZE transition state,and thus improve the catalytic efficiency against BZE.After site-directed mutagenesis,enzyme expression and purification,the catalytic activities of 5 designed mutants against BZE were determined by quantification of their reaction product benzoic acid（BA）using the HPLC-UV method.The results showed that A51L mutation（BZEase2）significantly enhanced the interaction between Coc E and BZE,and the K_Mwas decreased by 144 times（BZEase2,K_M=35.7μM;WT Coc E,K_M=5153μM）.Meanwhile,k_catwas increased approximately by 3-fold(BZEase2,k_cat=890 min^-1;WT Coc E,k_cat=301.2 min^-1),and the overall catalytic efficiency k_cat/K_Mhas been increased by 427 times.By mutating the key residue Ser117 in catalytic triad to Ala and removing the mutations causing cross-subunit disulfide bonds（L196C and I301C）,Coc E mutant（A51L/V116K/S117A/T172R/G173Q）was constructed and co-crystallized with BZE.The globular structure of Coc E mutant was obtained in complex with its product benzoic acid,which is probably due to S117A mutation in catalytic triad was not enough to abolish the catalytic activity thoroughly.This structure shows that there is no significant difference between Coc E mutants and the wildtype enzyme,and the key residual conformation in the activity center is consistent with our simulation results,providing supports for our computational calculation.2.BZE clearance by BZEase2 was characterized in Male Sprague Dawley rats.In this study,rats were firstly injected with saline,0.2 or 1 mg/kg of enzyme through tail vein after intravenous injection of 2 mg/kg BZE.～75μL of blood from saphenous veins was collected into heparin-treated capillary tubes at different time point after saline or enzyme injection,and immediately diluted with 100μL of 250μM paraoxon to terminate the enzymatic hydrolysis of BZE between blood sampling and HPLC analysis.The diluted blood samples were assayed for BZE and benzoic acid using the established HPLC-UV method.According to the in vivo data,BZEase2 dose-dependently accelerated BZE hydrolysis to benzoic acid and ecgonine.A single dose（1 mg/kg,iv）of BZEase2 could eliminate nearly all BZE in rats within only two minutes.3.The binding model of Coc E and ACh was constructed based on the structure of WT Coc E and the obtained crystal structure of Coc E-BA.The reaction mechanism of ACh hydrolysis catalyzed by Coc E was studied by molecular dynamics simulations and free energy calculation.The deacylation process was determined to be the rate-limiting step of the reaction.Based on the differences in the molecular charge distribution and net charge of ACh and BZE,13 mutants were designed on the basis of the quintuple mutant BZEase1 by reconstruct the electrostatic environment of the active site.The catalytic activities of these mutants against BZE and acetylthiochoine（ATC,a commonly used substrates for evaluating cholinesterase activity）were determined for preliminary screening.Results suggest when Ala51,a residual does not directly interact with substrate ACh,is mutated into positively charged amino acids,such as A51R（BZEase3）and A51K（BZEase4）,significantly reduce the catalytic efficiency of Coc E for ACh.The cholinesterase activity of BZEase3 and BZEase4 at 37℃was 1022 and 1328 times lower than that of WT Coc E.Although their catalytic efficiency against BZE is lower than BZEase2,they still show 44-fold and 39-fold improvement than WT Coc E,respectively.At physiological concentrations of BZE and ACh,the relative reaction rates for hydrolysis of BZE and ACh by these three mutants were significantly increased by 14622-fold,27139-fold,and 34977-fold compared to WT Coc E,respectively.All of above data suggests that these mutants dramatically improve the substrate specificity for BZE over ACh,and the cholinergic effect of BZE-metabolizing enzyme was significantly reduced.Conclusion:Through the iterative optimization strategy based on protein structure elucidation-catalytic mechanism study-rational design,the rationally designed Coc E mutant A51L/V116K/T172R/G173Q/L196C/I301C（BZEase2）had a 427-fold higher efficiency for BZE hydrolysis than that of WT Coc E.A single dose of BZEase2 could eliminate nearly all BZE in rats within two minutes,which provides a better starting point for the further development of BZE-metabolizing enzymes for complete elimination of cocaine-associated toxicity.By introducing A51R and A51K mutation to reconstruct the electrostatic environment in the active center,the binding affinity between enzyme and ACh was significantly decreased,and thus dramatically enhanced the substrate selectivity.Designed BZEase3 and BZEase4 showed over 1000-fold lower catalytic efficiency than WT Coc E,under the physiological concentrations of BZE and ACh their reaction rate for BZE hydrolysis were～30000-fold higher than that for ACh hydrolysis,indicating that these mutants have extremely high substrate selectivity for BZE than ACh,and their cholinergic side effects are negligible.This study focuses on re-engineering of Coc E to improve its enzymatic property from catalytic activity and substrate selectivity aspects,and the effort leads to the discovery of efficient and selective BZEase candidates for complete elimination of cocaine-associated toxicity.