ELECTROSTATIC ATTRACTION BY SURFACE-CHARGE DOES NOT CONTRIBUTE TO THECATALYTIC EFFICIENCY OF ACETYLCHOLINESTERASE

Acetylcholinesterases (AChEs) are characterized by a high net negative charge and by an uneven surface charge distribution, giving rise to a negative electrostatic potential extending over most of the molecular surface. To evaluate the contribution of these electrostatic properti es to the catalytic efficiency, 20 single-and multiple-site mutants of human AChE were generated by replacing up to seven acidic residues, v icinal to the rim of the active-center gorge (Glu84, Glu285, Glu292, A sp349, Glu358, Glu389 and Asp390), by neutral amino acids. Progressive simulated replacement of these charged residues results in a gradual decrease of the negative electrostatic potential which is essentially eliminated by neutralizing six or seven charges. In marked contrast to the shrinking of the electrostatic potential, the corresponding mutat ions had no significant effect on the apparent bimolecular rate consta nts of hydrolysis for charged and noncharged substrates, or on the K-i value for a charged active center inhibitor. Moreover, the k(cat) val ues for all 20 mutants are essentially identical to that of the wild t ype enzyme, and the apparent bimolecular rate constants show a moderat e dependence on the ionic strength, which is invariant for all the enz ymes examined. These findings suggest that the surface electrostatic p roperties of AChE do not contribute to the catalytic rate, that this r ate is probably not diffusion-controlled and that long-range electrost atic interactions play no role in stabilization of the transition stat es of the catalytic process.