The fluorescence of either N-methylacridinium (I) or 1 -methyl-7-hydroxyquinolinium (II) is totally quenched on binding to the catalytic site of acetylcholinesterase. Equilibrium titrations of 1 1S acetylcholinesterase at 0.1 M ionic strength with I confirmed previous reports that binding shows high specificity for the catalytic site. Analogous titrations with II indicated that only the protonated, cationic form of II binds and that binding has a specificity and stoichiometry similar to that of I. Under most of the experimental conditions introduced here, the reaction of either I or II with acetylcholinesterase was characterized by a single relaxation time. The bimolecular association constants for the reaction were unusually high, at 23 °C and ~0.1 M ionic strength; for I, k12 = 1.18 ± 0.03 × 9 M-1 s-1; for II, k12 = 2.18 ±0.15 × 109M-1 s-1. These constants were obtained from observed relaxation times both by a conventional analysis of equilibrium reactant concentrations and by a new method introduced here in which only the total ligand concentration need be known. At relatively high concentrations of enzyme and I, a second relaxation was observed; analyses of relaxation amplitudes indicated that this relaxation reflected independent ligand binding at a second, peripheral site on the enzyme. It has recently been suggested by M. Eigen that certain specific ligands may have unusually high bimolecular association constants with their target macromolecules because they can bind initially to peripheral sites and proceed to the specific site by surface diffusion on the macromolecule. A test of this proposal for acetylcholinesterase and I was conducted by introducing 30 mM Ca2+ to the solvent. No supporting evidence was obtained. Nevertheless, this proposal, applied to other sites with very low ligand affinities, may still partially account for the high bimolecular association rate constants.
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