AbstractThe 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 (Asp428β)—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 Thr474β(MADH)/Thr172β(AADH) to the catalytic carboxylate group and the nonconserved active site residue Tyr471β(MADH)/Phe169β(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.