Effective Charge on Acetylcholinesterase Active Sites Determined from the Ionic Strength Dependence of Association Rate Constants with Cationic Ligands

The reaction of the specific fluorescent cationic ligand N-methylacridinium with the active site of 11S acetylcholinesterase from electric eel was monitored by temperature-jump relaxation kinetics at a variety of ionic strengths. The ionic strength dependence of the bimolecular association rate constant is analyzed with a Brønsted-Debye-Hückel expression and leads to estimates of the association rate constant at zero ionic strength of k₁₂° = 1.1 × 10¹⁰M⁻¹s⁻¹at 25 °C and the net charge number of the enzyme active site of Z_E = -6.3. The ionic strength dependence of the second-order hydrolysis rate constant k_cat/ k_app for acetylthiocholine under steady-state conditions is also very pronounced and indicates a value of Z_E = -9. Thus, a large effective negative charge on the enzyme active site appears to be a general characteristic of its interaction with cationic ligands. The ionic strength dependence of k_cat/ k_app is identical with that of sodium chloride, sodium phosphate, and sodium citrate, thus ruling out any possibility that the phenomena arise from a specific, partially competitive binding of Na⁺ to the enzyme active site. Substitution of the calculated electrostatic parameters into theoretical equations indicates that the most significant effect of these Z_E values is a 2-3 order of magnitude reduction in the rate constant for dissociation of the initial ligand-enzyme encounter complex; this decrease renders the bimolecular reaction diffusion controlled. The high value of k₁₂⁰ and the space requirements of six to nine charged groups suggest that regions of the enzyme surface area larger than the catalytic sites themselves are effective in trapping cationic ligands.