Computational Study of the pK_aValues of Potential Catalytic Residues in the Active Site of Monoamine Oxidase B

Abstract

Monoamine oxidase (MAO), which exists in two isozymic forms, MAO A and MAO B, is an important flavoenzyme responsible for the metabolism of amine neurotransmitters such as dopamine, serotonin and norepinephrine. Despite extensive research effort, neither the catalytic nor the inhibition mechanisms of MAO have been completely understood. There has also been dispute with regard to the protonation state of the substrate upon entering the active site, as well as the identity of residues that are important for the initial deprotonation of irreversible acetylenic inhibitors, in accordance with the recently proposed mechanism. Therefore, in order to investigate features essential for the modes of action of MAO, we have calculated pK a values of three relevant tyrosine residues in the MAO B active site, with and without dopamine bound as the substrate (as well as the pK a of the dopamine itself in the active site). The calculated pK a values for Tyr188, Tyr398 and Tyr435 in the complex are found to be shifted upwards to 13.0, 13.7 and 14.7, respectively, relative to 10.1 in aqueous solution, ruling out the likelihood that they are viable proton acceptors. The altered tyrosine pK a values could be rationalized as an interplay of two opposing effects: insertion of positively charged bulky dopamine that lowers tyrosine pK a values, and subsequent removal of water molecules from the active site that elevates tyrosine pK a values, in which the latter prevails. Additionally, the pK a value of the bound dopamine (8.8) is practically unchanged compared to the corresponding value in aqueous solution (8.9), as would be expected from a charged amine placed in a hydrophobic active site consisting of aromatic moieties. We also observed potentially favorable cation-π interactions between -NH 3 + group on dopamine and aromatic moieties, which provide stabilizing effect to the charged fragment. Thus, we offer here theoretical evidence that the amine is most likely to be present in the active site in its protonated form, which is similar to the conclusion from experimental studies of MAO A (Jones et al. J. Neural Trans. 2007, 114, 707-712). However, the free energy cost of 3 transferring the proton from the substrate to the bulk solvent is only 1.9 kcal mol -1 , leaving open the possibility that the amine enters the chemical step in its neutral form. In conjunction with additional experimental and computational work, the data presented here should lead towards a deeper understanding of mechanisms of the catalytic activity and irreversible inhibition of MAO B, which can allow for the design of novel and improved MAO B inhibitors.

How are Biogenic Amines Metabolized by Monoamine Oxidases?

“…We truncated the enzyme to the flavin moiety of the FAD cofactor (isoalloxazine group 4a ) and three tyrosine side‐chains ( p ‐hydroxytoluenes of Tyr188, Tyr398 and Tyr435), which all form the hydrophobic “aromatic cage”,6,33 an important structural feature of MAO enzymes. Previously, we calculated the p K a values of the Tyr residues with bound dopamine, employing the full dimensionality of the protein,34 and obtained an upward shift to 13.0–14.7 (10.1 in aqueous solution). This clearly confirmed the hydrophobic nature of the active site, which indicates that gas‐phase calculations on truncated MAOs are quite reliable.…”

Revealing Monoamine Oxidase B Catalytic Mechanisms by Means of the Quantum Chemical Cluster Approach

“…27,28 Despite of the ongoing discussion regarding alternative mechanisms in MAO, it would seem unlikely that such closely related enzymes would employ different mechanistic strategies to accomplish very similar amine oxidations. 21 In fact, to carry out such a task two different active site electrostatic preorganizations would have to occur in order to stabilize transition states involving a carbanion in MAO A or a carbocation in MAO B. In this context, the Quantum Chemical Cluster Approach can be used as a robust tool with the aim of rationalising the accumulated experimental evidence for these enzymes.…”

Anionic Phenolic Compounds Bind Stronger with Transthyretin than Their Neutral Forms: Nonnegligible Mechanisms in Virtual Screening of Endocrine Disrupting Chemicals

“…60,61 If the pK a values in the binding site are used in the modeling, the resulting QSAR model can be different. Currently, it is a challenge to calculate the pK a values in the binding site, which needs further studies.…”