Kinetic and molecular modelling analyses of butyrylcholinesterase activity toward 'inverse' substrates

The high catalytic power of cholinesterases results from an efficient interplay between optimised structure, molecular dynamics and fine adjustment of substrates in the active site. Kinetic and molecular modelling investigations of the catalysis of 'inverse' ester substrates compared to 'normal' ester substrates by butyrylcholinesterase (BChE) were performed to shed light on the effect of isomeric inversion of the ester bond on binding and catalytic steps. The substrates used were phenyl-acetate (PhA), -propionate (PhPr), -butyrate (PhBu). The 'inverse' substrates used were benzoic acid methyl ester (BAME), phenyl acetic acid methyl ester (PAAME) and phenyl propionic acid methyl ester (PPAME). Inversion of the ester bond dramatically affected catalytic efficiency. Comparative analysis of the kinetics of both types of substrates showed that hydrolysis of 'inverse' substrates was only possible at high enzyme concentrations, and the rates were first-order, indicating (S)_max << K_m. The ratio of specific activity (k_cat/K_m) between normal and 'inverse' substrates increased 12-fold from PhA/BAME to PhPr/PAAME. PhBu was the best substrate, while PPAME was not hydrolysed by BChE. Thus, introduction of methylene group(s) in the acyl moiety of substrates progressively increased k_cat/K_m, while introduction of methylene group(s) in phenol/alcohol moiety of 'inverse' substrate considerably decreased k_cat/K_m, indicating dramatic loss of substrate complementarity to the active site. QM/MM and molecular dynamics simulations revealed the molecular basis for altered kinetics of 'inverse' substrate hydrolysis. The observed effects stemmed from reduced stability of reaction intermediates and transition states, along with inversion of the productive complex conformation. BChE exhibited no selectivity for this conformation or alternative binding modes.