Analysis of the reaction of carbachol with acetylcholinesterase using thioflavin T as a coupled fluorescence reporter

Abstract

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acylated enzyme intermediate is produced. Carbamates are very poor substrates that, like other AChE substrates, form an initial enzyme-substrate complex with free AChE (E) and proceed to an acylated enzyme intermediate (EC), which is then hydrolyzed. However, the hydrolysis of EC is slow enough to resolve the acylation and deacylation steps on the catalytic pathway. Here, we focus on the reaction of carbachol (carbamoylcholine) with AChE. The kinetics and thermodynamics of this reaction are of special interest because carbachol is an isosteric analogue of the physiological substrate acetylcholine. We show that the reaction can be monitored with thioflavin T as a fluorescent reporter group. The fluorescence of thioflavin T is strongly enhanced when it binds to the P-site of AChE, and this fluorescence is partially quenched when a second ligand binds to the A-site to form a ternary complex. Analysis of the fluorescence reaction profiles was challenging because four thermodynamic parameters and two fluorescence coefficients were fitted from the combined data both for E and for EC. Respective equilibrium dissociation constants of 6 and 26 mM were obtained for carbachol binding to the A- and P-sites in E and of 2 and 32 mM for carbachol binding to the A- and P-sites in EC. These constants for the binding of carbachol to the P-site are about an order of magnitude larger (i.e., indicating lower affinity) than previous estimates for the binding of acetylthiocholine to the P-site.

SCHEME 1

Figure 1

Structures of acetylcholine analogs and thioflavin T.

SCHEME 2

SCHEME 3

SCHEME 4

SCHEME 5

Figure 2

Reactions of carbachol with AChE monitored by thioflavin T fluorescence. Reactions were initiated by stopped-flow mixing of AChE with carbachol and thioflavin T as outlined in the Experimental Procedures, and the fluorescence F was recorded continuously. Initial concentrations of thioflavin T (15.6 μM) and AChE (152 nM) were the same in each reaction, while the respective concentrations of carbachol were 0, 0.5, 10, 40, and 60 mM (from the upper to the lower trace taken at time t = 0). F_B (eq 5) from the components individually was 83.

Figure 3

Analysis of a reaction of carbachol with AChE. Reaction traces were obtained in the presence of thioflavin T under the conditions in Figure 2 with 1.67 mM carbachol in Panel A or 60 mM carbachol in Panel B. Traces were analyzed in SigmaPlot both with the one-component, single-exponential fit in eq 6 and the two-component, double exponential fit in eq A2 in the Appendix to obtain values of F_EM, F_ECN, and k₁₂ + k₂₁. The solid red line shows the two-component fit and the dotted green line indicates the one-component fit. Respective values of k₁₂ + k₂₁ for the overall one- and two-component fits were 5.94 ± 0.02 and 6.53 ± 0.02 min⁻¹ in Panel A and 23.8 ± 0.3 and 27.0 ± 0.4 min⁻¹ in Panel B. In the two-component fits, k₁₂₂ = 0.68 ± 0.04 in Panel A and 4.3 ± 0.3 in Panel B.

Figure 4

Determination of carbachol binding constants with free AChE from the initial fluorescence of AChE-bound thioflavin T. From reference (23). F_EM values from mixtures of AChE (70 - 155 nM), thioflavin T (1.0 - 15.6 μM, as noted), and the indicated concentration of carbachol were determined from stopped-flow traces as in Figure 3. The F_EM data from each set at fixed thioflavin T concentration were fitted initially to eq 10 to obtain f_EL for the set, and F_EM/f_EL from all three sets were then analyzed simultaneously with eq 10 as described in the Experimental Methods. Parameters obtained from the data fitting are shown in Table 1. The dotted lines show the one-site fit when carbachol is assumed to have no affinity for the P-site ((iK_S)⁻¹ fixed at 0) with fitted parameters K_M+S = 6.2, f_EL/f_ESL = 1.85, and i₂ =2.1.

Figure 5

Determination of carbachol binding constants with carbamoylated AChE from the fluorescence of AChE-bound thioflavin T after the carbachol reactions have reached a final steady state. F_ECN values from mixtures of AChE (70 - 155 nM), thioflavin T (1.0 - 15.6 μM, as noted), and the indicated concentration of carbachol were determined from stopped-flow traces as in Figure 3. The F_ECN and then F_ECN/f_ECL data were analyzed with eq 11 as described for F_EM and then F_EM/f_EL in Figure 4. Parameters obtained from the data fitting are shown in Table 2. The dotted lines show the fit when carbachol is assumed to bind to only one site, (e.g., carbachol and thioflavin T compete for the P-site: (i_ECK_ECS)⁻¹ and i_EC2⁻¹ fixed at 0 with fitted K_M+S = 57).