Structure‐Reactivity Relationships as Probes for the Inhibition Mechanism of Cholesterol Esterase by Aryl Carbamates. I. Steady‐State Kinetics

For substituted phenyl‐ N ‐butyl carbamates (1) and 4‐nitrophenyl‐ N ‐substituted carbamates (2), linear relationships between values of NH proton chemical shift (δ NH ), p Ka , and log k [OH] and Hammett substituent constant (σ) or Taft substituent constant (σ*) are observed. Carbamates 1 and 2 are pseudo‐substrate inhibitors of porcine pancreatic cholesterol esterase. Thus, the mechanism of the reaction necessitates that the inhibitor molecule and the enzyme form the enzyme‐inhibitor tetrahedral species at the K i step of the reaction and then form the carbamyl enzyme at the k c step of the reaction. Linear relationships between the logarithms of K i and k c for cholesterol esterase by carbamates 1 and σ are observed, and the reaction constants (ρs) are −3.4 and −0.13, respectively. Therefore, the above reaction forms the negative‐charge tetrahedral species and follows the formation of the relatively neutral carbamyl enzymes. For the inhibition of cholesterol esterase by carbamates 2 except 4‐nitrophenyl‐ N ‐phenyl carbamate and 4‐nitrophenyl‐ N‐t ‐butyl carbamate, linear relationships of ‐log K i and log k c with σ* are observed and the ρ* values are −0.50 and 1.03, respectively. Since the above reaction also forms the negative‐charge tetrahedral intermediate, it is possible that the K i step of this reaction is further divided into two steps. The first K i step is the development of the positive‐charge at the carbamate nitrogen from the protonation of the carbamate nitrogen. The second K i step is the formation of the tetrahedral intermediate with the negative‐charge at the carbonyl oxygen. From Arrhenius plots of a series of inhibition reactions by carbamates 1 and 2, the isokinetic and isoequilibrium temperatures are different from the reaction temperature (25°C). Therefore, the observed ρ and ρ* values only depend upon the electronic effects of the substituents. Taken together, the cholesterol esterase inhibition mechanism by carbamates 1 and 2 is proposed.