Hydrogen tunnelling in enzyme-catalysed H-transfer reactions: flavoprotein and quinoprotein systems

Abstract

It is now widely accepted that enzyme-catalysed C-H bond breakage occurs by quantum mechanical tunnelling. This paradigm shift in the conceptual framework for these reactions away from semi-classical transition state theory (TST, i.e. including zero-point energy, but with no tunnelling correction) has been driven over the recent years by experimental studies of the temperature dependence of kinetic isotope effects (KIEs) for these reactions in a range of enzymes, including the tryptophan tryptophylquinone-dependent enzymes such as methylamine dehydrogenase and aromatic amine dehydrogenase, and the flavoenzymes such as morphinone reductase and pentaerythritol tetranitrate reductase, which produced observations that are also inconsistent with the simple Bell-correction model of tunnelling. However, these data-especially, the strong temperature dependence of reaction rates and the variable temperature dependence of KIEs-are consistent with other tunnelling models (termed full tunnelling models), in which protein and/or substrate fluctuations generate a configuration compatible with tunnelling. These models accommodate substrate/protein (environment) fluctuations required to attain a configuration with degenerate nuclear quantum states and, when necessary, motion required to increase the probability of tunnelling in these states. Furthermore, tunnelling mechanisms in enzymes are supported by atomistic computational studies performed within the framework of modern TST, which incorporates quantum nuclear effects.

Figure 1

Representation of the model for the hydrogen transfer reaction used to interpret the experimental data (see text and references Benkovic & Hammes-Schiffer (2003), Kohen & Klinman (1998) and Kohen & Klinman (1999) for more details). The three axes are: E, energy; q, environmental coordinate (from which the transferred hydrogen atom is excluded) and HC, hydrogen coordinate. The four vertical panels show the potential energy curve as a function of HC for three values of the environmental coordinate: q^R is for the reactant, q^* is for the transition state and q^P is for the product. The grey spheres represent the ground state vibrational wave function of the hydrogen nucleus. The panel labelled M shows a Marcus-like view of the free-energy curves as functions of this environmental coordinate. The motions of the environment modulate the symmetry of the double well, thus allowing the system to reach a configuration with (nearly) degenerate quantum states (q=q^*), from which the hydrogen is able to tunnel (F.C. Term in equation (3.1)). The difference between panels a and b is a gating motion that reduces the distance between the two wells along the HC-axis (r_H) away from its equilibrium value (r₀). This motion increases the probability of tunnelling at the (nearly) degenerate configuration q^* (active dynamics term in equation (3.1)).

Figure 2

(a) The structure of the tryptophan tryptophylquinone (TTQ) cofactor. (b) Reaction mechanism of the reductive half-reaction of MADH. Steps enclosed in the box represent binding steps. A similar scheme has been proposed for the reaction of AADH with aromatic primary amines. The tunnelling step is denoted k₃.

Figure 3

Schematic of the active site in methylamine dehydrogenase, illustrating the QM/MM partition used in the calculations and the mechanism corresponding to the tunnelling reaction. The QM region is shown on a white background and part of the surrounding MM region on a grey background, with link atoms circled; hydrogen bonds are depicted by dashed lines.

Figure 4

Energetics along the reaction path for proton tunnelling. Potential energy relative to reactant (solid black line), and minimum energy path (including zero-point energy; solid grey line) calculated to obtain the KIEs for proton transfer. The reaction coordinate, s, is the mass-weighted difference in position from the transition state (1 bohr=0.529 Å), with s=0 at the top of the barrier and negative at the reactant side.

Scheme 1

Proposed scheme for the oxidative half-reaction of morphinone reductase. The identity of the proton donor in the oxidative half-reaction is not known.