Kinetic characterization of a cocaine hydrolase engineered from mouse butyrylcholinesterase

Mouse butyrylcholinesterase (mBChE) and an mBChE-based cocaine hydrolase (mCocH, i.e. the A(199)S/S(227)A/S(287)G/A(328)W/Y(332)G 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 A(199)S/F(227)A/S(287)G/A(328)W/Y(332)G mutant). It has been demonstrated that mCocH and hCocH have improved the catalytic efficiency of mBChE and hBChE against (-)-cocaine by similar to 8- and similar to 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 (k(cat)/K-M) 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 similar to 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.