Actylcholinesterase (AChE) catalyzes the hydrolysis of acetylcholine (ACh) in nerve synapses. It was proposed that AChE's large negative charge and dipole moment serve to attract the positively charged ACh substrate down a long, narrow gorge to the active site (Sussman, et al. (1991) Science 253, 872-9; Ripoll et al. (1993) PNAS-USA 90, 5128-32). Yet in site-directed mutagenesis experiments, in which several negative charged residues near the gorge entrance were neutralized, catalytic activity was not greatly affected (Shafferman, et al. (1994) EMBO-J. 13, 3448-55).
Electrostatic calculations were done on the X-ray structure of Torpedo californica AChE and on homology built models of native human AChE and some mutant AChEs, in which a number of acidic residues on the surface near the catalytic gorge, or near the base of the gorge, were neutralized. The dipole moments due to the fixed atomic charges, the potentials along the narrow catalytic gorge and the average potential in hemispheric regions surrounding the gorge and opposite sites of the enzyme were calculated, and the effect of ionic strength on T. californica AChE's electrostatic properties was examined.
Both native AChEs have a large dipole moment oriented roughly parallel to the gorge axis and a constantly increasing negative potential inside the gorge itself. The gorge potential is relatively little affected by such external factors as ionic strength above 0.01 M and neutralizing up to 7 surface acidic residues near the gorge entrance, although these factors did change significantly the overall dipole and external potential above the gorge. These same factors had little effect on catalytic parameters. In contrast, replacing a single acidic residue near the gorge base adversely affects both the gorge potential and catalysis. Our calculations suggest that the large negative charge and dipole moment of these cholinesterases serve mainly to draw the positively charged substrate towards the enzyme and the entrance of the gorge. Once inside, however, these factors are superseded by an independent, gradually increasing potential that helps to draw the positively acetylcholine substrate toward the active site.