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The fluorescence of either N-methylacridinium (I) or 1 -methyl-7-hydroxyquinolinium (11) is totally quenched on binding to the catalytic site of acetylcholinesterase. Equi- librium titrations of 11s acetylcholinesterase at 0.1 M ionic strength with I confirmed previous reports that binding shows high specificity for the catalytic site. Analogous titrations with 11 indicated that only the protonated, cationic form of I1 binds and that binding has a specificity and stoichiometry similar to that of I. Under most of the experimental conditions intro- duced here, the reaction of either I or I1 with acetylcholinest- erase was characterized by a single relaxation time. The bi- molecular association constants for the reaction were unusually high,at23 "Cand-0.1 Mionicstrength;f orI,klz= 1.18f 0.03 X IO9 M-' s-l; for 11, k12 = 2.18 f 0.15 X lo9 M-ls-l. These constants were obtained from observed relaxation times both by a conventional analysis of equilibrium reactant con- The speed with which acetylcholinesterase catalyzes the hydrolysis of acetylcholine has long been appreciated. Studies by Michel and Krop (1951), Lawler (1961), Kremzner and Wilson (1964) and others are in quite close agreement on a maximum turnover number (kcat) of 1.6 X lo4 s-l at pH 8 and 25 "C (Rosenberry and Bernhard, 1971; Rosenberry, 1975a). Recently it has been emphasized (Rosenberry, 1975a) that this enzymatic hydrolysis is also extremely efficient at the perhaps more physiologically relevant acetylcholine concentrations below the apparent Michaelis constant Kapp (<0.1 mM). At these concentrations the appropriate rate parameter is the second-order rate constant kcat/Kapp. Values of kcat/Kapp for acetylcholine and several other cationic substrates are around 2 X IO8 M-' 5-l at 25 "C and 0.1 M ionic strength (Rosen- berry, 1975a) and thus approach a limit generally expected for diffusion-controlled enzyme reactions (Eigen and Hammes, 1963). In agreement with this suggestion, a low deuterium oxide isotope effect of 1.1 is associated with kcat/Kapp for acetylcholine (Rosenberry, 1975b); this observation indicates that the rate-limiting step for acetylcholine hydrolysis at low substrate concentrations precedes general acid-base catalysis and is likely to involve either the bimolecular reaction step or a subsequent conformational change of the enzyme-substrate complex (Rosenberry, 1975b). Because of the importance of the initial steps associated with the interaction of acetylcholinesterase with specific ligands, we have used temperature-jump relaxation kinetics to inves- tigate the reactions of this enzyme with N-methylacridinium

Interaction of ligands with acetylcholinesterase. Use of temperature-jump relaxation kinetics in the binding of specific fluorescent ligands