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

The fluorescence of either N-methylacridinium ( I ) or 1 -methyl-7-hydroxyquinolinium (11) is totally quenched on binding to the catalytic site of acetylcholinesterase. Equilibrium titrations of 11s acetylcholinesterase a t 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 introduced here, the reaction of either I or I1 with acetylcholinesterase was characterized by a single relaxation time. The bimolecular association constants for the reaction were unusually h igh ,a t23 "Cand-0.1 Mionics t rength ; for I ,k lz= 1 . 1 8 f 0.03 X I O 9 M-' s-l; for 11, k 1 2 = 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 conT h e 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 I O 8 M-' 5-l a t 25 "C and 0.1 M ionic strength (Rosenberry, 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 a t 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 investigate the reactions of this enzyme with N-methylacridinium + troin the Departments of Neurology and Biochemistry, College of Ph>\ ic ian \ and Surgeons, Columbia University, New York, New York IOO?? . and the Max-Planck-lnstitut fuer Biochemie, D-8033 Martinsried bci bluenchen, West Germany. Receired December 14. 1976. This i n vchtigation w a s wpported, in part. by United States Public Health Service (;rant NUS-03304. National Science Foundation Grant PCM73-00744. and 'I Y.4TO Senior Fellowship in Science to T.L.R. centrations 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 a t 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 m M 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. (1) and I-methyl-7-hydroxyquinolinium (11). Both I and the zwitterionic form of 11 are highly fluorescent, and the fluorescence of both I and I1 is totally quenched when they bind