Analysis of Classical and Quantum Paths for Deprotonation of Methylamine by Methylamine Dehydrogenase

Abstract

The hydrogen-transfer reaction catalysed by methylamine dehydrogenase (MADH) with methylamine (MA) as substrate is a good model system for studies of proton tunnelling in enzyme reactions--an area of great current interest--for which atomistic simulations will be vital. Here, we present a detailed analysis of the key deprotonation step of the MADH/MA reaction and compare the results with experimental observations. Moreover, we compare this reaction with the related aromatic amine dehydrogenase (AADH) reaction with tryptamine, recently studied by us, and identify possible causes for the differences observed in the measured kinetic isotope effects (KIEs) of the two systems. We have used combined quantum mechanics/molecular mechanics (QM/MM) techniques in molecular dynamics simulations and variational transition state theory with multidimensional tunnelling calculations averaged over an ensemble of paths. The results reveal important mechanistic complexity. We calculate activation barriers and KIEs for the two possible proton transfers identified-to either of the carboxylate oxygen atoms of the catalytic base (Asp428beta)-and analyse the contributions of quantum effects. The activation barriers and tunnelling contributions for the two possible proton transfers are similar and lead to a phenomenological activation free energy of 16.5+/-0.9 kcal mol(-1) for transfer to either oxygen (PM3-CHARMM calculations applying PM3-SRP specific reaction parameters), in good agreement with the experimental value of 14.4 kcal mol(-1). In contrast, for the AADH system, transfer to the equivalent OD1 was found to be preferred. The structures of the enzyme complexes during reaction are analysed in detail. The hydrogen bond of Thr474beta(MADH)/Thr172beta(AADH) to the catalytic carboxylate group and the nonconserved active site residue Tyr471beta(MADH)/Phe169beta(AADH) are identified as important factors in determining the preferred oxygen acceptor. The protein environment has a significant effect on the reaction energetics and hence on tunnelling contributions and KIEs. These environmental effects, and the related clearly different preferences for the two carboxylate oxygen atoms (with different KIEs) in MADH/MA and AADH/tryptamine, are possible causes of the differences observed in the KIEs between these two important enzyme reactions.

Substrate binding to mammalian 15-lipoxygenase

“…Therefore, our simulations show that the two 15-rLO-1 physiological substrates (AA and LA) can lead to different proton donor-acceptor distances when forming complexes with 15-rLO-1, in such a way that a significant change in the corresponding KIEs could be expected, as observed experimentally. However, the final effect of the difference observed in the dynamical fluctuations and d(H-OH) distances on the reaction rate and the KIEs are not easy to predict a priori [24][25][26]. This is especially true when the hydrogen transfer is dominated by vibration-driven tunneling mechanism, as it is suspected to be the case here and as it has been proven for soybean lipoxygenase-1 [27].…”

Optimal control design of laser pulses for mode specific vibrational excitation in an enzyme–substrate complex

“…in enzymes. The proton transfer in the reaction of tryptamine catalysed by AADH has been investigated in detail with QM/ MM methods [16,17], as have similar enzymes such as methylamine dehydrogenase [20,21]. The rate-determining step has been shown to be the transfer of a proton from a saturated carbon atom to a nearby (carboxylate) oxygen [16].…”

“…3 shows the partitioning between the QM (shaded) and MM parts of the system. The B3LYP method provides a good description of the proton transfer reaction, and yields potential energy and dipole moment surfaces of good quality [20,32].…”

Nuclear quantum effects and kinetic isotope effects in enzyme reactions

“…A non-exhaustive list of such enzyme studies includes dihydrofolate reductase (DHFR) [41,46,56,90,102,[110][111][112][113][114], alcohol dehydrogenase (ADH) [50,83,115,116], lipoxygenase [55,59,[117][118][119], methylamine dehydrogenase [115,[120][121][122][123][124], thymidylate synthase [125,126], chorismate mutase [127][128][129], several decarboxylases [43,100,101,130], methylmalonyl-CoA mutase [131,132], alanine racemase [39,100,133], formate dehydrogenase [42,134], nitroalkane oxidase [8,108,135], morphinone reductase [116], and glyoxalase [134,136]. In the following we will focus on some examples of work performed in our group, with which ...…”