Isotope Effects on Enzyme-Catalyzed Acyl Transfer from <i>p</i>-Nitrophenyl Acetate:  Concerted Mechanisms and Increased Hyperconjugation in the Transition State

Abstract

To examine the mechanism of enzymatic acyl transfers from p -nitrophenyl acetate (PNPA), isotope effects were measured for the reaction of PNPA with chymotrypsin, carbonic anhydrase, papain, and Aspergillus acid protease. The isotope effects were measured at the β-deuterium ( D k ), carbonyl carbon ( 13 k ), carbonyl oxygen ( 18 k carbonyl ), leaving group phenolic oxygen ( 18 k lg ), and leaving group nitrogen ( 15 k ) positions. D k ranged from 0.982 ± 0.002 to 0.999 ± 0.002. 13 k ranged from 1.028 ± 0.002 to 1.036 ± 0.002. 18 k carbonyl ranged from 1.0064 ± 0.0003 to 1.007 ± 0.001. 18 k lg ranged from 1.141 ± 0.0002 to 1.330 ± 0.0007. 15 k ranged from 0.9997 ± 0.0007 to 1.0011 ± 0.0002. Uncatalyzed acyl transfer from PNPA to oxygen and sulfur nucleophiles proceeds by a concerted mechanism. All of the enzymatic reactions showed isotope effects consistent with a concerted mechanism like that seen in uncatalyzed aqueous reactions, but exhibited smaller inverse β-deuterium isotope effects than seen in the nonenzymatic aqueous reactions. This phenomenon may be explained by greater hydrogen bonding or electrostatic interaction with the ester carbonyl group in enzymatic transition states relative to nonenzymatic aqueous transition states. Quantum mechanical calculations were used to estimate the magnitude of changes in hyperconjugation and C−H bond order due to protonation of a carbonyl oxygen.