Isotope Effects on Enzyme-Catalyzed AcylTransfer from p-Nitrophenyl Acetate: Concerted Mechanisms and Increased Hyperconjugation inthe Transition State

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Journal of the American Chemical Society

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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 (Dk), carbonyl carbon (13k), carbonyl oxygen (18kcarbonyl), leaving group phenolic oxygen (18klg), and leaving group nitrogen (15k) positions. Dk ranged from 0.982 ± 0.002 to 0.999 ± 0.002. 13k ranged from 1.028 ± 0.002 to 1.036 ± 0.002. 18kcarbonyl ranged from 1.0064 ± 0.0003 to 1.007 ± 0.001. 18klg ranged from 1.141 ± 0.0002 to 1.330 ± 0.0007. 15k 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.

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