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

Abstract

AbstractFor substituted phenyl‐N‐butyl carbamates (1) and 4‐nitrophenyl‐N‐substituted carbamates (2), linear relationships between values of NH proton chemical shift (δNH), pKa, and logk[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 Ki step of the reaction and then form the carbamyl enzyme at the kc step of the reaction. Linear relationships between the logarithms of Ki and kc 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 ‐logKi and logkc 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 Ki step of this reaction is further divided into two steps. The first Ki step is the development of the positive‐charge at the carbamate nitrogen from the protonation of the carbamate nitrogen. The second Ki 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.