A Model Reaction Assesses Contribution of H-Tunneling and Coupled Motions to Enzyme Catalysis

Abstract

To assess the contribution of physical features to enzyme catalysis, the enzymatic reaction has to be compared to a relevant uncatalyzed reaction. While such comparisons have been conducted for some hydrolytic and radical reactions, it is most challenging for biological hydride transfer and redox reactions in general. Here, the same experimental tools used to study the H-tunneling and coupled motions for enzymatic hydride transfer between two carbons were used in the study of an uncatalyzed model reaction. The enzymatic oxidations of benzyl alcohol and its substituted analogues mediated by alcohol dehydrogenases were compared to the oxidations by 9-phenylxanthylium cation (PhXn(+)). The PhXn(+) serves as an NAD(+) model, while the solvent, acetonitrile, models the protein environment. Experimental comparisons included linear free energy relations with Hammett reaction constant (ρ) of zero versus -2.7; temperature-independent versus temperature-dependent primary KIEs; deflated secondary KIEs with deuteride transfer (i.e., primary-secondary coupled motion) versus no coupling between secondary KIEs and H- or D-transfer; and large versus small secondary KIEs for the enzymatic versus uncatalyzed alcohol oxidation. Some of the differences may come from differences in the order of microscopic steps between the catalyzed versus uncatalyzed reactions. However, several of these comparative experiments indicate that in contrast to the uncatalyzed reaction the transition state of the enzymatic reaction is better reorganized for H-tunneling and its H-donor is better rehybridized prior to the C-H→C transfer. These findings suggest an important role for these physical features in enzyme catalysis.

Computational Replication of the Abnormal Secondary Kinetic Isotope Effects in a Hydride Transfer Reaction in Solution with a Motion Assisted H-Tunneling Model

“…28 Additionally, the rehybridization of the donor and acceptor carbons are found to be nonsynchronous with that in alcohol (from sp 3 to sp 2.60 ) lagging behind that in Xn + (from sp 2 to sp 2.53 ). These results suggest a TRS with imbalanced development in charge and geometry in the alcohol moiety and nonsynchronous rehybridization between the two reacting carbons.…”

“…47 The quantitative comparison between the two systems manifests the role of protein motions in such catalysis: protein motions advance the geometry change of the system, providing favorable orbital conditions for H-tunneling to occur. 28,29 …”

“…However, even if there is other evidence demonstrating H-tunneling, some solution and enzymatic H-transfers still show no 1° isotope effect on 2° KIEs. 7,25−28 Furthermore, we recently reported a deflated 2° KIE and no 1° isotope effect on 2° KIEs for a solution hydride transfer reaction that cannot be explained by the traditional theories. 29 In that work, semi-classical models predicted KIEs close to the equilibrium isotope effect (EIE) for a late TS, but the measured KIEs were closer to unity (typical of an early TS).…”

Imbalanced tunneling ready states in alcohol dehydrogenase model reactions: rehybridization lags behind H-tunneling

“…This is opposite to the order found in enzymes, strongly supporting the above important proposal regarding the role of protein motions in catalysis. 24 In order to strengthen the conclusion, it is necessary to expand the study to other H-transfer systems in solution, especially those in which the 21 KIEs on both the H-donor and H-acceptor can be determined so that the rehybridization of both can be examined. For this purpose, use of the xanthylium ion (Xn + ) as a hydride acceptor is necessary since the 21 KIE on its 9-C-L bond (sp 2 to sp 3 ) can also be determined.…”

“…[24][25][26][27] Here, kinetics of the hydride-transfer reactions from alcohols to Xn + ClO 4 À in MeCN were similarly determined by following the decay of the Xn + UV-Vis absorption (Fig. S1, ESIw).…”