Isotope Effects on Enzyme-Catalyzed Acyl Transfer from p-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.

Kinetic and Mechanistic Characterization of the Formyl-CoA Transferase from Oxalobacter formigenes

“…It is also possible that attack of the Asp 169 carboxylate on formyl-CoA proceeds via a concerted mechanism in which an oxyanion intermediate is not formed. We note that heavy atom kinetic isotope effect measurements (56) and theoretical studies of acyl transfer from thioesters (57) provide evidence for such a proposal. The resolution of this question, however, will likely require the use of 13 C and 18 O isotope effect measurements.…”

The catalytic cycle of β-lactam synthetase observed by x-ray crystallographic snapshots

“…5). For acyl substitution reactions involving very good leaving groups, as is the case here, concerted mechanisms are believed to occur through a tetrahedral transition state (20,21), or even by a dissociative mechanism and formation of an acylium ion (22,23). For nitrogen nucleophiles, however, the development of positive charge on nitrogen as bond formation to the carbonyl carbon proceeds (Fig.…”

The Use of Isotope Effects to Determine Enzyme Mechanisms

“…This isotope effect results from decreased hyperconjugation in the tetrahedral transition state and was 4 -5% inverse for attack by oxygen nucleophiles (35). However, in the enzymatic reactions this value was 0 -2% inverse (36). These data show that in the enzymatic reactions the enzymes polarize the carbonyl group and increase hyperconjugation when the substrate binds (Fig.…”

“…Al Hengge and Rob Hess carried out a thorough study of the reactions of p-nitrophenyl acetate with various nucleophiles (35) and with several enzymes (36). In opposition to what most textbooks say, these reactions do not have a tetrahedral intermediate but are concerted.…”