Enzymatic H-Transfer Requires Vibration-Driven Extreme Tunneling

Abstract

Enzymatic breakage of the substrate C-H bond by Methylophilus methyltrophus (sp. W3A1) methylamine dehydrogenase (MADH) has been studied by stopped-flow spectroscopy. The rate of reduction of the tryptophan tryptophylquinone (TTQ) cofactor has a large kinetic isotope effect (KIE = 16.8 +/- 0.5), and the KIE is independent of temperature. Analysis of the temperature dependence of C-H bond breakage revealed that extreme (ground state) quantum tunneling is responsible for the transfer of the hydrogen nucleus. Reaction rates are strongly dependent on temperature, indicating thermally induced, vibrational motion drives the H-transfer reaction. The data provide direct experimental evidence for enzymatic bond breakage by extreme tunneling driven by vibrational motion of the protein scaffold. The results demonstrate that classical transition state theory and its tunneling derivatives do not adequately describe this enzymatic reaction.

Efficient estimators for quantum instanton evaluation of the kinetic isotope effects: Application to the intramolecular hydrogen transfer in pentadiene

“…However, unexpectedly large KIE's have recently been observed also in several enzymatic reactions at physiological temperatures. 2,3 These experiments imply that in some biochemical reactions, the hydrogen transfer proceeds via tunneling even at physiological temperatures, often with the help of thermally excited "promoting" vibrations that modulate the distance between the hydrogen donor and acceptor. These quantum effects have also been observed in several numerical simulations.…”

Interpretation of Kinetic Isotope Effects in Enzymatic Cleavage of Carbon-Hydrogen Bonds

“…Scrutton and coworkers [11,12] have studied the oxidative demethylation of primary amines to form aldehydes and ammonia by methylamine dehydrogenase (MADH) and also by aromatic amine dehydrogenase (AADH), which shows much the same behavior. In these reactions a proton is abstracted by an active-site base from an iminoquinone intermediate while an electron is transferred to a tryptophylquinone cofactor.…”

“…Our earlier efforts [1,7] to fit the data to a tunneling model were unsuccessful. Our present analysis starts with the basic substrate methylamine [11], for which a KIE of 16 (ln ¼ 2:8) was observed. Application of Eq.…”

“…If the data pass this test, qualitative and quantitative information can be obtained on the mechanism of the tunneling reaction and the structure of the reaction center. In subsequent sections the model is applied to a representative set of kinetic data on enzymatic systems and models thereof, including soybean lipoxygenase-1 (SLO1) [5,6], coenzyme B 12 [9,10], primary amine dehydrogenases [11,12], and phenylalanine hydroxylase [13].…”

Three applications of path integrals: equilibrium and kinetic isotope effects, and the temperature dependence of the rate constant of the [1,5] sigmatropic hydrogen shift in (Z)-1,3-pentadiene

“…While this often is an appropriate point of view, it fails completely in some cases, and almost always when hydrogen is involved in bond breaking or formation. Strong nuclear quantum effects have been observed in many reactions and recently even in several enzymatic reactions [1][2][3]. In such situations, one should treat some or all nuclei quantum mechanically.…”