ISOTOPE EFFECTS ON ENZYME-CATALYZED ACYL TRANSFER FROM P-NITROPHENYL ACETATE - CONCERTED MECHANISMS AND INCREASED HYPERCONJUGATION IN THE TRANSITION-STATE

To examine the mechanism of enzymatic acyl transfers from p-nitropheny l acetate (PNPA), isotope effects were measured for the reaction of PN PA with chymotrypsin, carbonic anhydrase, papain, and Aspergillus acid protease. The isotope effects were measured at the beta-deuterium ((D )k), carbonyl carbon ((13)k), carbonyl oxygen ((18)k(carbonyl)), leavi ng 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) ran ged 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 +/- 0.00 07 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 concerte d mechanism like that seen in uncatalyzed aqueous reactions, but exhib ited smaller inverse P-deuterium isotope effects than seen in the none nzymatic aqueous reactions. This phenomenon may be explained by greate r hydrogen bonding or electrostatic interaction with the ester carbony l group in enzymatic transition states relative to nonenzymatic aqueou s transition states. Quantum mechanical calculations were used to esti mate the magnitude of changes in hyperconjugation and C-H bond order d ue to protonation of a carbonyl oxygen.