ELECTROSTATIC INFLUENCE ON THE KINETICS OF LIGAND-BINDING TO ACETYLCHOLINESTERASE - DISTINCTIONS BETWEEN ACTIVE-CENTER LIGANDS AND FASCICULIN

To explore the role that surface and active center charges play in ele ctrostatic attraction of ligands to the active center gorge of acetylc holinesterase (AChE), and the influence of charge on the reactive orie ntation of the ligand, we have studied the kinetics of association of cationic and neutral ligands with the active center and peripheral sit e of AChE, Electrostatic influences were reduced by sequential mutatio ns of six surface anionic residues outside of the active center gorge (Glu-84, Glu-91, Asp-280, Asp-283, Glu-292, and Asp-372) and three res idues within the active center gorge (Asp-74 at the rim and Glu-202 an d Glu-450 at the base), The peripheral site ligand, fasciculin 2 (FAS2 ), a peptide of 6.5 kDa with a net charge of +4, shows a marked enhanc ement of rate of association with reduction in ionic strength, and thi s ionic strength dependence can be markedly reduced by progressive neu tralization of surface and active center gorge anionic residues, By co ntrast, neutralization of surface residues only has a modest influence on the rate of cationic m-trimethylammoniotrifluoroacetophenone (TFK) association with the active serine, whereas neutralization of residu es in the active center gorge has a marked influence on the rate but w ith little change in the ionic strength dependence, Brownian dynamics calculations for approach of a small cationic ligand to the entrance o f the gorge show the influence of individual charges to be in quantita tive accord with that found for the surface residues, Anionic residues in the gorge may help to orient the ligand for reaction or to trap th e ligand. Bound FAS2 on AChE not only reduces the rate of TFK+ reactio n with the active center but inverts the ionic strength dependence for the cationic TFK+ association with AChE, Hence it appears that TFK+ m ust traverse an electrostatic barrier at the gorge entry imparted by t he bound FAS2 with its net charge of +4.