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Taurine/α-ketoglutarate dioxygenase is one of the most studied α-ketoglutarate-dependent dioxygenases (αKGDs), involved in several biotechnological applications. We investigated the key step in the catalytic cycle of the αKGDs, the hydrogen transfer process, by a quantum mechanics/molecular mechanics approach (B3LYP/CHARMM22). Analysis of the charge and spin densities during the reaction demonstrates that a concerted mechanism takes place, where the H atom transfer happens simultaneously with the electron transfer from taurine to the FeO cofactor. We found the quantum tunneling of the hydrogen atom to increase the rate constant by a factor of 40 at 5 °C. As a consequence, a quite high kinetic isotope effect close to 60 is obtained, which is consistent with the experimental value. [ABSTRACT FROM AUTHOR]

The flavin-containing enzyme monoamine oxidase (MAO) is essential for the enzymatic decomposition of amine neurotransmitters. The exact mechanism of the oxidative deamination of amines to aldehydes by the enzyme has not yet been fully understood despite extensive research on the area. The rate limiting step is the reductive half-reaction where the Hα together with two electrons of the amine substrate is transferred to the flavin cofactor. However, it is still not known whether the hydrogen is transferred as a proton or a hydride. Experimental results cannot be fully explained by either of those mechanisms. In our previous work, theoretical results based on QM/MM calculations of the full enzyme show an intermediate situation between these two cases. In this paper, we report on an in-depth computational analysis concerning the role of the enzymatic environment for the reaction mechanism of human MAO-B with different p-substituted benzylamines as substrates. Our results show that steric and electrostatic effects from the active site environment turn the mechanism closer to an asynchronous polar nucleophilic mechanism. We found indications that the protein environment of MAO-A enhances the polar nucleophilic character of the mechanism compared to that of MAO-B.

The flavoenzyme monoamine oxidase (MAO) is essential for the enzymatic decomposition of neurotransmitters. While it is commonly accepted that the rate limiting step of the reaction is the stereoselective abstraction of a hydrogen from the substrate, the precise mechanism is unknown. We modeled the reaction of human MAO-B with benzylamine by means of QM/MM calculations based on density functional theory. Oxidation of the unprotonated substrate was found to proceed with rates in good agreement with experimental values, while the protonated substrate does not react at room temperature. Our results support a concerted asynchronous polar nucleophilic mechanism. The lone pair of the amine-nitrogen interacts with a carbon atom of the flavin cofactor. During the reaction, this lone pair, as well as a proton, are transferred to the cofactor. Analysis of the electronic structure during the reaction rules out a radical mechanism.

The role of quantum mechanical atom tunneling during the conversion of glutamate to methylaspartate catalyzed by glutamate mutase is investigated by quantum mechanical/molecular mechanical (QM/MM) simulations based on coupled cluster and density functional calculations. The use of instanton theory allows us to calculate the tunneling contributions of up to 78 atoms in the active site. We calculate kinetic isotope effects (KIEs) and compare them to experimental data. The simulations lead to deuterium KIEs of 10 for the hydrogen abstraction from glutamate substrate and 16 for the hydrogen abstraction from methylaspartate substrate, which are consistent with the experimental results. The hydrogen abstraction from methylaspartate has higher primary deuterium and tritium (46.1) KIEs than the abstraction from glutamate. The tunneling effect increases the reaction rate by a factor of 12.3 for the hydrogen abstraction from methylaspartate at 0. Tunneling is supported by the environment by preparing the enzyme through classical motions. Consideraton of the tunneling contributions of more and more atoms around the active center shows that the motions at the ribose ring play a central role during the tunneling enhancement of the hydrogen transfers. Our simulations give new insight into the catalytic process in glutamate mutase and the way enzymes use tunneling effects for a successful catalysis.