Does Electrostatic Attraction or Steering by Charged Residues within the Gorge Contribute to the Reactivity of AChE?

Abstract

Studies with mutant AChEs where up to seven surface negative charges were neutralized demonstrated that electrostatic attraction does not contribute to the catalytic efficiency of the enzyme [1,2]. Yet, electrostatic steering due to the negatively charged residues in the gorge (D74 at its rim and E202 and E450 near its bottom) remained difficult to assess [3, 4]. We therefore extended the studies of these mutants with isosteric pairs of charged and noncharged substrates (ATC- acetylthiocholine; 3, 3-dimethylbutyl thioacetate-TB), charged and noncharged phosphate inhibitors (echothiophate, S-3,3-di-methylbutyl diethylthiophosphate) as well as with the transition state analog m-trimethylammonio trifluoroacetophenone (TFK). Replacements of D74 by negatively charged (D74E) positively charged (D74K) or neutral (D74N, D74G) residues resulted in small and uniform increase in values of Km for ATC except for D74K where the increase was much larger. On the other hand, the bimolecular rates of reactions with echothiophate and TFK were affected much more in cases of D74N and D74K than in those of D74E and E74G. All these mutations have almost no effect on catalytic activity toward the non-charged substrate, yet a small increase of phosphorylation rates by the noncharged OP inhibitor was observed for enzymes with noncharged residues at position 74 but not for D74K or D74E. These findings, together with the observation that ionic strength had small and equivalent effect on reactivities of all these enzymes, indicate that D74 probably does not enhance reactivity by steering charged ligands into the gorge but rather influences indirectly specific interactions of charged ligands with elements of the active center.