Kinetic characterization of a cocaine hydrolase engineered from mouse butyrylcholinesterase

Xiabin Chen, Xiaoqin Huang, Liyi Geng, Liu Xue, Shurong Hou, Xirong Zheng, Stephen Brimijoin, Fang Zheng, Chang Guo Zhan

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

Mouse butyrylcholinesterase (mBChE) and an mBChE-based cocaine hydrolase (mCocH, i.e. the A199S/S227A/S287G/A328W/Y332G mutant) have been characterized for their catalytic activities against cocaine, i.e. naturally occurring (-)-cocaine, in comparison with the corresponding human BChE (hBChE) and an hBChE-based cocaine hydrolase (hCocH, i.e. the A199S/F227A/S287G/A328W/Y332G mutant). It has been demonstrated that mCocH and hCocH have improved the catalytic efficiency of mBChE and hBChE against (-)-cocaine by ∼8- and ∼2000-fold respectively, although the catalytic efficiencies of mCocH and hCocH against other substrates, including acetylcholine (ACh) and butyrylthiocholine (BTC), are close to those of the corresponding wild-type enzymes mBChE and hBChE. According to the kinetic data, the catalytic efficiency (kcat/KM) of mBChE against (-)-cocaine is comparable with that of hBChE, but the catalytic efficiency of mCocH against (-)-cocaine is remarkably lower than that of hCocH by ∼250-fold. The remarkable difference in the catalytic activity between mCocH and hCocH is consistent with the difference between the enzyme-(-)-cocaine binding modes obtained from molecular modelling. Further, both mBChE and hBChE demonstrated substrate activation for all of the examined substrates [(-)-cocaine, ACh and BTC] at high concentrations, whereas both mCocH and hCocH showed substrate inhibition for all three substrates at high concentrations. The amino-acid mutations have remarkably converted substrate activation of the enzymes into substrate inhibition, implying that the rate-determining step of the reaction in mCocH and hCocH might be different from that in mBChE and hBChE.

Original languageEnglish (US)
Pages (from-to)243-251
Number of pages9
JournalBiochemical Journal
Volume466
DOIs
StatePublished - Mar 1 2015

Keywords

  • Cholinesterase
  • Drug abuse
  • Enzyme
  • Hydrolysis
  • Kinetics

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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