Structure‐Reactivity Relationships as Probes to Acetylcholinesterase Inhibition Mechanisms by Aryl Carbamates. I. Steady‐State Kinetics

From enzyme kinetics, 4‐nitrophenyl‐ N ‐substituted carbamates 1 are characterized as pseudo‐substrate inhibitors of acetylcholinesterase. However, the activity of the carbamyl enzyme does not recover in the presence of a competitive inhibitor, edrophonium. Therefore, carbamates 1 should be called as the “pseudo‐pseudo‐substrate” inhibitors of the enzyme. Moreover, the ‐log K i , log k c , and log k i values are linearly correlated with Taft‐Ingold equation, log ( k/k o ) = ρ*σ* + δ E s . A three‐step AChE inhibition mechanism by carbamates 1 is proposed. The first step is the pre‐equilibrium protonations of carbamates 1 with ρ* value of −1.4 from p K a ‐σ*‐correlation. The second step is the enzyme‐carbamates 1 tetrahedral intermediate formation from nucleophilic attack of the active site Ser200 on the protonated carbamates 1 . The ρ* value for the ‐log K i ‐σ*‐ E s ‐correlation indicates that the true ρ* value for the second step is 0.5 [= −0.9 ‐ (‐1.4)]. The δ value of 0.56 for the ‐log K i ‐σ*‐ E s ‐correlation indicates that carbamates 1 with bulky substituents retarded the formation of enzyme‐inhibitor tetrahedral intermediates. The third step ( k c step) is the carbamylation step and is the carbamyl enzyme conjugate formation from the enzyme‐carbamates 1 tetrahedral intermediate. The ρ* value of 0.21 for the log k c ‐correlation indicates that the transition state for the carbamylation step is more negative charge than the enzyme‐carbamates 1 tetrahedral intermediate. Moreover, the k c step is insensitive to substituent effects since there is a cancellation of electronic demands for bond‐making and bond‐breaking components, like S N 2 reactions. The δ value of 0.00 for the log k c ‐correlation indicates that the k c step is independent of substituent steric effect. Therefore, the product of this step carbamyl enzyme conjugate is as crowded as the enzyme‐carbamates 1 tetrahedral intermediate and is likely bound to the leaving group, p ‐nitrophenol.