Theoretical and experimental investigations of electrostatic effects on acetylcholinesterase catalysis and inhibition

The role of electrostatics in the function of acetylcholinesterase (AChE) h as been investigated by both theoretical and experimental approaches. Secon d-order rate constants (k(E) = k(cat)/K-m) for acetylthiocholine (ATCh) tur nover have been measured as a function of ionic strength of the reaction me dium for wild-type and mutant AChEs. Also, binding and dissociation rate co nstants have been measured as a function of ionic strength for the respecti ve charged and neutral transition state analog inhibitors m -(N,N,N-trimeth yiammonio)trifluoroacetophenone (TMTFA) and m-(t-butyl)trifluoroacetophenon e (TBTFA). Linear free-energy correlations between catalytic rate constants and inhibition constants indicate that k(E) for ATCh turnover is rate limi ted by terminal binding events. Comparison of binding rate constants for TM TFA and TBTFA attests to the sizable electrostatic discrimination of AChE. Free energy profiles for cationic ligand release from the active sites of w ild-type and mutant AChEs have been calculated via a model that utilizes th e structure of T. californica AChE, a spherical ligand, and energy terms th at account for electrostatic and van der Waals interactions and chemical po tential. These calculations indicate that EA and EI complexes are not bound with respect to electrostatic interactions, which obviates the need for a 'back door' for cationic ligand release. Moreover, the computed energy barr iers for ligand release give linear free-energy correlations with log(k(E)) for substrate turnover, which supports the general correctness of the comp utational model. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved .