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 "pseudopseudo-substrate" inhibitors of the enzyme. Moreover, the -logKi, logk c , and logk i values are linearly correlated with Taft-Ingold equation, log (k/k o ) = r*s* + d 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 r* value of -1.4 from pK a-s*-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 r* value for the -logKi-s*-Es-correlation indicates that the true r* value for the second step is 0.5 [= -0.9 -(-1.4)]. The d value of 0.56 for the -logKi-s*-Es-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 r* value of 0.21 for the logkc-correlation indicates that the transition state for the carbamylation step is more negative charge than the enzyme-carbamates 1 tetrahedral intermediate. Moreover, the kc step is insensitive to substituent effects since there is a cancellation of electronic demands for bond-making and bond-breaking components, like SN2 reactions. The d value of 0.00 for the logkc-correlation indicates that the kc 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.

4-Nitrophenyl-N-substituted carbamates (1) are characterized as pseudosubstrate inhibitors of acetylcholinesterase. The first step is formation of the enzyme-inhibitor tetrahedral intermediate with the inhibition constant (Ki), the second step is formation of the carbamyl enzyme with the carbamylation constant (kc), and the third step is hydrolysis of the carbamyl enzyme with decarbamylation constant (kd). According to pre-steady state kinetics the Ki step is divided further into two steps: (1) formation of the enzyme-inhibitor complex with the dissociation constant (KS) and (2) formation of the enzyme-inhibitor tetrahedral intermediate from the complex with the equilibrium constant (k2/k-2). Since the inhibitors are protonated in pH 7.0 buffer solution, the virtual dissociation constant (KS') of the enzyme-protonated inhibitor complex can be calculated from the equation, -log KS'=-log KS-pKa + 14. The -logKS, -log KS', log k2, and log k-2 values are multiply linearly correlated with the Jave equation (log(k/k0)=rho*sigma* + deltaEs + psi pi). For -log KS'-sigma*-Es)pi-correlation, the rho* value of -0.4 indicates that the enzyme-protonated inhibitor complexes have more positive charges than the protonated inhibitors, the delta value of 0.44 suggests that the bulkily substituted inhibitors lessen the reaction due to the difficulty of the inhibitors to enter the narrow enzyme active site gorge, and the psi value of 0.27 implies that the inhibitors with hydrophobic substituents accelerate the inhibitors entering the active site gorge of the enzyme. For log k2/k-2,-sigma*-Es-pi-correlation, the rho* value of 1.1 indicates that the enzyme-protonated inhibitor tetrahedral intermediates have more negative charges than the enzyme-protonated inhibitor complexes, the delta value of 0.15 suggests that the bulkily substituted inhibitors are difficult to bind into a small acyl binding site of the enzyme, and the psi value of -0.3 implies that the inhibitors with hydrophobic substituents resist binding to the hydrophilic acyl binding site of the enzyme.