Direct Kinetic Evidence for the Formation of an Acylpyridinium Intermediate in Synthetic p-Nitrophenyl Esterase-Catalyzed Hydrolysis Reactions.

The kinetics of the hydrolysis of p-nitrophenyl alkanoates 2 catalyzed by 1 were investigated in aqueous Tris buffer solution. Direct evidence for the existence of an N- acylpyridinium intermediate 3 was obtained for 1-catalyzed hydrolysis of 2 n2-10. Increase of the alkyl chain length leads to an increase in the acylation rate which reaches a maximum for 2 n6. The acylation rate then decreases progressively with further increases of alkyl chain length in substrate esters. The deacylation rate was also found to exhibit a maximum for the same substrate 2 n6. These results are similar to those previously reported with cholesterol esterase as catalyst for the same hydrolysis reaction. The acylation reaction is first-order in catalyst concentration and exhibits saturation kinetics at high substrate concentration in accordance with the Michaelis-Menten model for enzyme reaction kinetics.