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.