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 -logK i , logk c , and logk 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 pKa-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 -logK 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 -logK 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 logk 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 logk 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.