Decarbamoylation of acetylcholinesterases is markedly slowed as carbamoyl groups increase in size

Abstract

Carbamates are esters of substituted carbamic acids that react with acetylcholinesterase (AChE) by initially transferring the carbamoyl group to a serine residue in the enzyme active site accompanied by loss of the carbamate leaving group followed by hydrolysis of the carbamoyl enzyme. This hydrolysis, or decarbamoylation, is relatively slow, and half-lives of carbamoylated AChEs range from 4 min to more than 30 days. Therefore, carbamates are effective AChE inhibitors that have been developed as insecticides and as therapeutic agents. We show here, in contrast to a previous report, that decarbamoylation rate constants are independent of the leaving group for a series of carbamates with the same carbamoyl group. When the alkyl substituents on the carbamoyl group increased in size from N-monomethyl- to N,N-dimethyl-, N-ethyl-N-methyl-, or N,N-diethyl-, the decarbamoylation rate constants decreased by 4-, 70-, and 800-fold, respectively. We suggest that this relationship arises as a result of active site distortion, particularly in the acyl pocket of the active site. Furthermore, solvent deuterium oxide isotope effects for decarbamoylation decreased from 2.8 for N-monomethylcarbamoyl AChE to 1.1 for N,N-diethylcarbamoyl AChE, indicating a shift in the rate-limiting step from general acid-base catalysis to a likely conformational change in the distorted active site.

Keywords: Acetylcholinesterase; Alzheimer's disease; Decarbamoylation.

Figure 1.

Molecular structures of carbamoyl and leaving groups in carbamates examined in this report.

Figure 2.

Reactions of the carbamates a) N-monomethyl neostigmine and b) rivastigmine with hAChE. The carbamates or buffer blanks were mixed with hAChE in a stopped-flow apparatus and reactions were monitored at 412 nm with 0.25 mM acetylthiocholine and 2.0 mM DTNB as outlined in the Experimental Procedures (black traces labeled “observed”). The reaction data for carbamates were fitted to Eq. 2 (pink traces labeled “fit”) with R set to zero, either t₀ or A_412(t0) fixed at a value determined by inspection or from controls without enzyme, and with v₀, A_412(t0), k₁₂ and k₂₁ as the fitted parameters. The reaction data for blanks without carbamate gave a linear fit in panel A (cyan trace labeled “fit”) or a linear fit in panel B that was calculated by normalizing panel A data to the AChE concentration (cyan trace labeled “calculated”). a) Final concentrations of N-monomethyl neostigmine and hAChE after mixing were 5.0 μM and 2.0 nM, respectively. Fitting to Eq. 2 with t₀ fixed at 0.100 min gave k₁₂ = 14 min⁻¹ and k₂₁ = 13 × 10⁻³ min⁻¹. b) Final concentrations of rivastigmine and hAChE after mixing were 2.0 mM and 0.33 nM, respectively. Fitting to Eq. 2 with A_412(t0) fixed at 0.114 min gave k₁₂ = 1.24 min⁻¹ and k₂₁ = 3.1 × 10⁻³ min⁻¹. The carbamate trace in panel b was offset by 0.5 min.

Figure 3.

Decarbamoylation reactions following treatment of hAChE with a) N-monomethyl neostigmine and b) rivastigmine. AChE was radiomethylated, carbamoylated, isolated on a spin column, and subjected to continuous spectrophotometric assay at 412 nm with 0.25 mM acetylthiocholine and 0.33 mM DTNB as outlined in the Experimental Procedures (black traces labeled “observed”). The reaction data were fitted to Eq. 2 (pink traces labeled “fit”) with k₁₂ and t₀ set to zero, v_f fixed at a predetermined value, and A_412(t0), R, and k₂₁ as the fitted parameters. Calculated traces corresponding to v_f are shown in cyan (labeled “calculated”). a) Concentrations of N-monomethyl neostigmine and hAChE in the initial mix were 1.0 mM and 1.8 nM, respectively, and 50 μL of the spin column recovery was added to 3.0 mL of assay buffer containing 0.25 mM acetylthiocholine. Fitting to Eq. 2 with v_f fixed at 0.143 ΔA/min gave k₂₁ = (12.9 ± 0.02) × 10⁻³ min⁻¹ and R = 0.90. b) Concentrations of rivastigmine and hAChE in the initial mix were 2.0 mM and 1.8 nM, respectively, and 50 μL of the spin column recovery was added to 3.0 mL of assay buffer containing 0.25 mM acetylthiocholine. Fitting to Eq. 2 with v_f fixed at 0.192 ΔA/min gave k₂₁ = (155 ± 0.4) × 10⁻⁶ min⁻¹ and R = 0.996. The lower value of R in panel a) relative to panel b) reflects faster reactivation of N-monomethylcarbamoylated AChE during gel filtration and prior to initiation of the reaction trace. Carbamate traces in panels a) and b) were offset by 2.0 and 5.0 min, respectively.

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