Fluorescent probes of acetylcholinesterase

Mooser, Gregory; Schulman, Howard; Sigman, David S.

doi:10.1021/bi00759a008

Cited by 87 publications

(61 citation statements)

References 21 publications

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“…Our observations with N-methylacridinium are consistent with a previous report (Mooser et al, 1972) that the fluorescence of the ligand is totally quenched on binding to the enzyme (fluorescence of bound ligand do. 1% of fluorescence of free ligand).…”

Section: S C H E M E I X I ?supporting

confidence: 93%

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

Rosenberry¹,

Neumann²

1977

Biochemistry

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ABSTRACT: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 high,at23 "Cand-0.1 Mionicstrength;forI,klz= 1 . 1 8 f 0.03 X IO9 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 con-T 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 IO8 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 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 investigate the reactions of this enzyme with N-methylacridinium + troin the

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Section: S C H E M E I X I ?supporting

confidence: 93%

“…reasonable agreement with a value of -100 s-' estimated from dilution experiments involving N-methylacridinium and acetylcholinesterase (Mooser and Sigman, 1975).…”

Section: Tablfsupporting

confidence: 85%

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

Rosenberry¹,

Neumann²

1977

Biochemistry

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“…3B). Again, the association constant obtained for MAC with native AChE, (3.8 2 0.7).106 M", is similar to values reported earlier for Electrophorus AChE (Mooser et al, 1972). To estimate the decrease in affinity of the two ligands for the MG preparations of AChE, we generated theoretical titration curves corresponding to various decreases in the values of the association constants.…”

Section: Fluorescence Titration Of the Mg States With Inhibitors Of Achesupporting

confidence: 78%

“…The active site is located at the bottom of this gorge. Two fluorescent probes, propidium and N-methylacridinium (MAC), both of which are potent reversible inhibitors of AChE (Mooser et al, 1972;Taylor & Lappi, 1975), display high affinity for two different binding sites located, respectively, on the rim and at the bottom of the gorge, which are at least 10 8, apart (Eichler et al, 1994a). We considered it worthwhile to check whether the MG,,,, and MG,,, retained any capacity to bind these ligands, so as to clarify whether the secondary structural elements preserved in these MG states retained a native-like tertiary fold (Baldwin, 1991;Ritsyn, 1992Ritsyn, , 1995Chan et al, 1995).…”

Section: Fluorescence Titration Of the Mg States With Inhibitors Of Achementioning

confidence: 99%

“…In the case of MAC, the excitation and emission wavelengths were 360 nm and 460 nm, respectively, and in the case of propidium, 535 nm and 620 nm. For both ligands, the stoichiometry of binding to native enzyme was taken as one mole of ligand per catalytic subunit (Mooser et al, 1972;Taylor & Lappi, 1975). Accordingly, the titration curves were analyzed on the assumption that, at a given concentration of ligand, the fluorescence intensity can be expressed as:…”

Section: Fluorescence Titration With Propidium and Macmentioning

confidence: 99%

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Two partially unfolded states oftorpedo californicaacetylcholinesterase

et al. 1996

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Chemical modification with sulfhydryl reagents of the single, nonconserved cysteine residue CysZ3' in each subunit of a disulfide-linked dimer of Torpedo californica acetylcholinesterase produces a partially unfolded inactive state. Another partially unfolded state can be obtained by exposure of the enzyme to 1-2 M guanidine hydrochloride. Both these states display several important features of a molten globule, but differ in their spectroscopic (CD, intrinsic fluorescence) and hydrodynamic (Stokes radii) characteristics. With reversal of chemical modification of the former state or removal of denaturant from the latter, both states retain their physicochemical characteristics. Thus, acetylcholinesterase can exist in two molten globule states, both of which are long-lived under physiologic conditions without aggregating, and without either intraconverting or reverting to the native state. Both states undergo spontaneous intramolecular thioldisulfide exchange, implying that they are flexible. As revealed by differential scanning calorimetry, the state produced by chemical modification lacks any heat capacity peak, presumably due to aggregation during scanning, whereas the state produced by guanidine hydrochloride unfolds as a single cooperative unit, thermal transition being completely reversible. Sucrose gradient centrifugation reveals that reduction of the interchain disulfide of the native acetylcholinesterase dimer converts it to monomers, whereas, after such reduction, the two subunits remain completely associated in the partially unfolded state generated by guanidine hydrochloride, and partially associated in that produced by chemical modification. It is suggested that a novel hydrophobic core, generated across the subunit interfaces, is responsible for this noncovalent association. Transition from the unfolded state generated by chemical modification to that produced by guanidine hydrochloride is observed only in the presence of the denaturant, yielding, on extrapolation to zero guanidine hydrochloride, a high free energy barrier (ca. 23.8 kcal/mol) separating these two flexible, partially unfolded states.

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