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-substitute d carbamates (2), linear relationships between values of NH proton chemical shift (delta(NH)). pKa, and logk([OH]) and Hammett substituent constant (s igma) or Taft substituent constant (sigma*) are observed. Carbamates 1 and 2 are pseudo-substrate inhibitors of porcine pancreatic cholesterol esteras e. Thus, the mechanism of the reaction necessitates that the inhibitor mole cule 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 o f the reaction. Linear relationships between the logarithms of K-i and k(c) for cholesterol esterase by carbamates 1 and sigma are observed, and the r eaction constants (ps) are -3.4 and -0.13, respectively. Therefore, the abo ve reaction forms the negative-charge tetrahedral species and follows the f ormation of the relatively neutral carbamyl enzymes. For the inhibition of cholesterol esterase by carbamates 2 except 4-nitrophenyl-N-phenyl carbamat e and 4-nitrophenyl-N-t-butyl carbamate, linear relationships of -logK(i) a nd logk(c) with sigma* are observed and the rho* values are -0.50 and 1.03, respectively. Since the above reaction also forms the negative-charge tetr ahedral 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 th e positive-charge at the carbamate nitrogen from the protonation of the car bamate nitrogen. The second K-i step is the formation of the tetrahedral in termediate with the negative-charge at the carbonyl oxygen. From Arrhenius plots of a series of inhibition reactions by carbamates 1 and 2, the isokin etic and isoequilibrium temperatures are different from the reaction temper ature (25 degrees C). Therefore, the observed rho and rho* values only depe nd upon the electronic effects of the substituents. Taken together, the cho lesterol esterase inhibition mechanism by carbamates 1 and 2 is proposed.