Kinetics of acetylthiocholine binding to electric eel acetylcholinesterase in glycerol/water solvents of increased viscosity. Evidence for a diffusion-controlled reaction.

Steady-state kinetic studies were made on the very efficient enzyme hydrolysis of acetylthiocholine by electric eel acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) in glycerol/water solvents of increased viscosity. Determinations of the very fast minimum substrate association rate constants kmin, (2 . 10(8) M-1 . s-1 at I approximately 0.1 M and 25 degrees C) from the Michaelis parameters, V/(Km[E0]), were made at low substrate concentrations in order to obtain kmin directly. kmin was shown to be strongly dependent upon viscosity, which is characteristic of a diffusion-controlled reaction. kmin is as large or larger than plausible models for a simple diffusion-controlled reaction between a charged enzyme and substrate would suggest. Enhancement of the diffusion-controlled reaction through nonspecific binding of substrate to the highly negatively charged acetylcholinesterase followed by two-dimensional surface diffusion in a random walk to the active site may be a factor in this enzyme mechanism. Evidence for this comes from the viscosity dependence of kmin. Using the surface diffusion model it is estimated that the binding-site target area on acetylcholinesterase is effectively increased a minimum of 8-fold.