| Abstract
| - To examine the mechanism of enzymatic acyl transfers fromp-nitrophenyl acetate (PNPA), isotopeeffects were measured for the reaction of PNPA with chymotrypsin,carbonic anhydrase, papain, and Aspergillusacid protease. The isotope effects were measured at theβ-deuterium (Dk), carbonyl carbon(13k), carbonyloxygen (18kcarbonyl), leaving groupphenolic oxygen (18klg), and leavinggroup nitrogen (15k) positions.Dk rangedfrom 0.982 ± 0.002 to 0.999 ± 0.002.13k ranged from 1.028 ± 0.002 to 1.036± 0.002. 18kcarbonylrangedfrom 1.0064 ± 0.0003 to 1.007 ± 0.001.18klg ranged from 1.141 ±0.0002 to 1.330 ± 0.0007. 15krangedfrom 0.9997 ± 0.0007 to 1.0011 ± 0.0002. Uncatalyzed acyltransfer from PNPA to oxygen and sulfurnucleophiles proceeds by a concerted mechanism. All of theenzymatic reactions showed isotope effectsconsistent with a concerted mechanism like that seen in uncatalyzedaqueous reactions, but exhibited smallerinverse β-deuterium isotope effects than seen in the nonenzymaticaqueous reactions. This phenomenon maybe explained by greater hydrogen bonding or electrostatic interactionwith the ester carbonyl group in enzymatictransition states relative to nonenzymatic aqueous transition states.Quantum mechanical calculations wereused to estimate the magnitude of changes in hyperconjugation and C−Hbond order due to protonation of acarbonyl oxygen.
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