An analysis of reaction pathways for proton tunnelling in methylamine dehydrogenase

Abstract

Computational methods have now become a valuable tool to understand the way in which enzymes catalyse chemical reactions and to aid the interpretation of a diverse set of experimental data. This study focuses on the influence of the condensed-phase environment structure on proton transfer mechanisms, with an aim to understand how C-H bond cleavage is mediated in enzymatic reactions. We shall use a combination of molecular simulation, ab initio or semi-empirical quantum chemistry and semi-classical multidimensional tunnelling methods to consider the primary kinetic isotope effects of the enzyme methylamine dehydrogenase (MADH), with reference to an analogous application to triosephosphate isomerase. Analysis of potentially reactive conformations of the system, and correlation with experimental isotope effects, have highlighted that a quantum tunnelling mechanism in MADH may be modulated by specific amino acid residues, such as Asp428, Thr474 and Asp384.

Figure 1

The tryptophan tryptophylquinone (TTQ) cofactor in MADH.

Figure 2

The mechanism of the reductive half-reaction catalysed by MADH. The deprotonation step (3), outlined in the box, is the experimentally determined rate-limiting step.

Figure 3

The active site in MADH depicting a typical reactant configuration. The QM region used in the QM/MM calculations is shown unshaded with link atoms circled.

Figure 4

Potential energy barriers (filled triangles) and reaction energies (filled squares) (kcal mol⁻¹) for an ensemble of enzyme configurations with proton transfer to O2 of Asp428.

Figure 5

Plot of potential energy (kcal mol⁻¹), corresponding to conformation B, against (a) the change in the O₁–C₅, O₁–H₄ and C₅–H₄ distances (Å) for proton transfer to O1 and (b) the change in the O₂–C₅, O₂–H₄ and C₅–H₄ distances (Å) for proton transfer to O2.

Figure 6

Competing proton transfer mechanisms in MADH: (a) carbanion formation resulting in a classical reaction and (b) delocalization of the anion resulting in proton tunnelling (the arrows indicate significant changes in structure).

Figure 7

Carbanion formation and delocalization of anionic character during the proton transfer mechanism in MADH.

Figure 8

Active-site and proton transfer mechanism in TIM (QM region is shown unshaded).

Figure 9

Potential energy barriers, ΔV^≠, and reaction energies, Δ_rV, for proton transfer in an ensemble of enzyme configurations to O2 in MADH (filled diamonds) and TIM (filled squares). Five configurations corresponding to transfer to O1 in MADH are indicated by A–E (filled triangles).

Figure 10

Potential energy profile for proton transfer in MADH.