Examining Electrostatic Preorganization in Monoamine Oxidases A

Article pubs.acs.org/JPCB

Examining Electrostatic Preorganization in Monoamine Oxidases A and B by Structural Comparison and pKa Calculations Matej Repič,† Miha Purg,† Robert Vianello,‡ and Janez Mavri†,§,* †

Laboratory for Biocomputing and Bioinformatics, National Institute of Chemistry, Hajdrihova 19, SI−1000 Ljubljana, Slovenia Quantum Organic Chemistry Group, Ruđer Bošković Institute, Bijenička cesta 54, HR−10000 Zagreb, Croatia § EN−FIST Centre of Excellence, Dunajska 156, SI−1000 Ljubljana, Slovenia ‡

S Supporting Information *

ABSTRACT: Monoamine oxidases (MAO) A and B are important flavoenzymes involved in the metabolism of amine neurotransmitters. Orru et al. (J. Neural Transm. 2013, 120, 847−851) recently presented experimental results that have challenged the prevailing assumption that MAO A and MAO B employ an identical catalytic mechanism. We compared the spatial configuration of ionizable groups in both isozymes and estimated the time-averaged electrostatic potential by calculating the pKa values of five active site residues. Superimposition of both experimental structures shows very close overlap and the RMSD in placements of ionizable groups within 16 Å of the reaction center is only 0.847 Å. This similarity is also reflected in the calculated pKa values, where the largest difference between the MAO A and MAO B pKa values was found for residues Tyr188 in MAO B and the corresponding Tyr197 in MAO A assuming 1.23 units. The pKa values for the other four studied residues differ by less than 0.75 units. The results show that the electrostatic preorganizations in both active sites are very similar, supporting the idea that both enzymes work by the same mechanism.

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INTRODUCTION Monoamine oxidases (MAO) A and B are important flavoenzymes involved in the metabolism of amine neurotransmitters and play a pivotal role in the metabolism of serotonin and dopamine.1 While the two isoforms, MAO A and MAO B, feature more than 70% sequence identity, they differ in substrate and inhibitor specificities.2,3 MAO A is mainly responsible for the degradation of serotonin and is thus a therapeutic target for the treatment of depression.4 MAO B, in turn, is a target for the clinical management of Parkinson disease, as it regulates dopamine levels in the central nervous system.5 Inhibiting MAO may also have a significant neuroprotective effect, since the products of its catalytic reaction are hydrogen peroxide and substrate aldehydes, which contribute to oxidative stress in the cell.6−8 Despite extensive experimental9−11 and computational12−15 research efforts, the precise catalytic steps used by monoamine oxidases remain elusive. MAOs convert amines to the corresponding imines by transferring two electrons and two protons from the substrate to the enzyme FAD cofactor, thereby transforming the latter to its reduced FADH2 form. While this fact is widely accepted, the exact stepwise mechanism of the rate-limiting step is still under question. We have shown in a previous study16 that, in terms of the energetics of the reaction, the initial transfer of a hydride anion from the substrate α-CH group has a much lower activation free energy than alternative proposals involving a proton17 or a hydrogen atom abstraction.10 Until recently, all mechanistic proposals shared a common assumption that MAO A and MAO B function using the same mechanism. Studies © 2014 American Chemical Society

performed by Edmondson and co-workers have shown that in a series of para-substituted benzylamine analogues, the enzymatic reaction of human liver MAO A is faster for analogues with electron-withdrawing substituents, which is consistent with proton abstraction.17 An analogous study concerning bovine MAO B, however, showed no electronic influence of the substituent on the rate, but established a linear correlation with the Taft steric parameterthe rate was inversely proportional to the parameter.18 Orru et al. recently performed a study with human MAO B and found a Taft relationship that is unlike the one found for bovine MAO B and the inverse of human MAO A.19 In human MAO A, the electronic parameter of the Taft correlation is positive, while in MAO B it is negative. On this basis, Orru et al. have put forward the idea that MAO A functions via a polar nucleophilic mechanism involving a proton transfer in the rate-limiting step, whereas MAO B functions by a hydride mechanism. Recently, Kästner and co-workers performed a QM/MM study of benzylamine and MAO B that seems to suggest that the transfer of two electrons and a proton in the rate-limiting step is concerted, but asynchronous, in agreement with the polar nucleophilic mechanism,15 although they failed to provide evidence for the initial substrate−flavin adduct that was originally postulated to facilitate the above-mentioned proton transfer.17 One is tempted to conclude that the absence of the complex formation in Kästner’s study indirectly supports the Received: January 23, 2014 Revised: March 10, 2014 Published: March 29, 2014 4326

dx.doi.org/10.1021/jp500795p | J. Phys. Chem. B 2014, 118, 4326−4332

The Journal of Physical Chemistry B

Article

hydride transfer mechanism proposed by us.16 Moreover, Akyüz and Erdem utilized ONION QM/MM calculations, taking into account the full dimensionality of the enzyme, and confirmed the hydride mechanism for both MAOs but suggested what they termed as a “slightly different hydride transfer mechanism” among isoforms.20 Soon after that, Atalay and Erdem performed a computational study on a model system to convincingly demonstrate the prevailing feasibility of the hydride transfer reaction over the proton transfer reaction.21 Enzyme catalysis is the rate-enhancement of the enzymatic reaction relative to the corresponding reaction in aqueous solution.22 Computational studies have shown that the bulk of the catalytic power of enzymes originates from electrostatic preorganization of the active site.23−25 The essence of this fact is that, in water, solvent molecules must reorient during the course of a reaction due to polarization induced by a changing charge distribution. On the other hand, this energetic penalty is much smaller in an enzyme, as the enzyme provides an electrostatic environment that has evolved to optimally solvate the transition state and requires much less reorganization energy to adapt.26 A change in the protonation states of ionizable residues results in an altered electrostatic potential pattern in the enzyme. The associated change in the solvation free energy of the transition state manifests as pH dependence of the rate of enzymatic reactions. The pKa values of ionizable residues in the enzyme active site are an excellent probe for the electrostatic environment, since pKa is very sensitive to any structural change and the associated alteration in the electrostatic potential. MAO A and MAO B are 70% homologous and because of very good overall matching (RMSD equals 0.66 Å) of the superimposed X-ray structures, we expect that their electrostatic potential pattern in the active site will be quite similar and accompanied by a similarity in the pK a values of the corresponding residues. The main contribution to the pKa values is made by the free energy of the solvation of the charged species in the (de)protonation reaction. In contrast to the electrostatic potential comparison for a number of points in space, pKa values can also be determined experimentally. Several authors have claimed that the MAO active site is hydrophobic and is composed of aromatic and apolar residues, including two tyrosines and the FAD cofactor, which form an aromatic cage.13,27 Despite the fact that monoamines are predominantly present as monocations at a physiological pH of 7.4, a hydrophobic active site has been proposed to favor unprotonated substrates and, furthermore, every catalytic proposal to date has agreed that the substrate must be neutral for the reaction to take place.9−11 Active site pKa values are difficult to determine experimentally and, similarly, while experimental pH rate profiles can provide tremendous insight, it can be hard to conclusively determine the identity of residues whose protonation state is being affected. For a review on this topic please see ref 28. Although there are many experimental methods that enable determination of the overall titration curve of a protein, only a few spectroscopic techniques possess sufficient resolution to allow for the determination of pKa values of individual residues in a protein.29 For MAO enzymes, a lot of research efforts have been devoted to experimentally measuring pKa values, but only data for a few residues that are close to the surface of the enzyme have been obtained.30−34 In addition, pKa calculations

continue to provide a significant challenge to biomolecular simulations.35−38 In this article, we calculated the pKa values of a few ionizable residues in MAOs in order to compare the electrostatic potential pattern in the active sites of both isoforms. In both cases, neutral dopamine was docked in the active site. Wellconverged free energy calculations of the pKa values were performed using the MOLARIS program package in conjunction with an all-atom representation of the solvated enzymes.

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COMPUTATIONAL METHODS High resolution crystal structures of MAO A39 and MAO B40 were obtained from the Protein Data Bank (accession codes 2Z5X41 and 2XFN,42 respectively). The protein chain and the FAD cofactor were retained, while the inhibitor and water molecules were removed from the crystal structure. Only the A chain was kept in MAO B and the resulting monomer unit was superimposed and aligned in UCSF Chimera43 using the MatchMaker tool. The structures were aligned and superimposed so that the RMSD between the FAD residues in both enzymes was minimized. The carboxyl carbon in Asp and Glu, the amino nitrogen in Lys and the guanidino carbon in Arg were chosen as charged group centers and the distances between these centers and the N5 atom of FAD were measured in UCSF Chimera. In line with the general consensus that dopamine enters the active site in a neutral form, we docked a neutral dopamine molecule into the active site manually in a way suitable for the catalytic step. We chose only one substrate on purpose in order to avoid the effects associated with ligands of different sizes on the pKa values. pKa calculations were performed using the semimacroscopic protein dipole/Langevin dipole approach of Warshel and co-workers in its linear response approximation version (PDLD/S-LRA).37,44,45 To parametrize the charge distribution of oxidized FAD and dopamine, electrostatic potential-derived atomic charges were obtained on the optimized structures at the (PCM)/B3LYP/631G(d) level of theory in conjunction with the UFF radii as implemented in the Gaussian09 program.46 The essence of the PDLD/S-LRA pKa calculation is to convert the problem of evaluating a pKa in a protein to an evaluation of the change in solvation energy associated with moving the charge from water to the protein. One must consider the thermodynamic cycle described by the following equation: ΔG p(AH p → A p− + H w +) = ΔGw(AH w → A w− + H w +) w→p − w→p + ΔGsol (A ) − ΔGsol (AH)

where p and w denote protein and water, respectively. This equation can be rewritten for each ionizable residue i, as pKap, i = pKaw, i −

qi̅ 2.3RT

w→p (AHi → A i−) ΔΔGsol

where the ΔΔG term consists of the last two terms of the previous equation and qi is the charge of the ionized form of the given residue for an acid qi̅ = −1(q(AH) = 0, q(A−) = −1)

and for a base qi̅ = +1(q(AH) = + 1, q(A−) = 0) 4327



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Figure 1. Ionizable residues that have an ionizable group within 16 Å (mesh sphere) of the N5 atom of FAD. MAO A residues and residue names are shown in green and MAO B in gray. Centers of ionizable groups are shown in the sphere representation (carboxyl carbon atom in Asp and Glu, amino nitrogen atom in Lys, and guanidino carbon atom in Arg).

averaged over 50 ps of simulation with a 0.5 fs time step, giving rise to a total simulation time of 1 ns for the entire thermodynamic perturbation. The calculated pKa values are sensitive to the applied external dielectric constant during the simulations. The choice of the correct dielectric constant to describe the protein interior is a very complicated issue and has been the subject of heated debates over the years.51 In our work, we employed ε = 10−16, however, due to the focus on the relative difference between pKa values in MAO A and MAO B, the choice would not change the qualitative picture and is thus of lesser importance. All PDLD/S-LRA calculations were performed using the ENZYMIX force field and the MOLARIS simulation package.52

The pKa calculations are thus reduced to two free energy calculations in addition to the experimental value in aqueous solution. The first simulation calculates the free energy associated with the transfer of a neutral residue from the protein to water and the other calculates the free energy cost of transferring the charged residue from water to the protein. In addition to these two values one must know only the experimental pKa in aqueous solution to determine the free energy associated with ionizing the residue in water. Please consult the Supporting Information for further computational details. This approach calculates pKa shifts relative to the aqueous solution by taking into account the protein environment dependent stabilization effects of the charged species. This method has previously been successfully applied to a wide range of systems of biological relevance, such as the aquaporin channel,47 carbonic anhydrase,48 and the bovine pancreatic trypsin inhibitor.49 Both proteins studied here were first explicitly solvated using the surface constrained all atom solvent (SCAAS) model,50 employing a water grid with a radius of 20 Å around the investigated residue. Long-range interactions were treated using the local reaction field (LRF) approach.44 The resulting system was equilibrated by running a 250 ps molecular dynamics simulation using a 0.5 fs time step at 300 K. After that, we evaluated pKa values using the PDLD/S-LRA approach, employing full atomic charges by averaging the corresponding values over the results obtained for the 20 protein configuration windows, connecting charged and uncharged states, each

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RESULTS AND DISCUSSION Studies of the enzyme active site electrostatics give insights into the reaction mechanism. On the basis of structural similarity, it seems reasonable to assume that MAO A and MAO B isoforms function via one mechanism, rather than by two distinct mechanisms. However, Edmondson and co-workers have challenged this assumption by providing experimental results showing that human MAO A and MAO B exhibit inverse electronic Taft correlations for a series of benzylamine analogues.17,19 This has opened up the possibility that the two isozymes function by two distinct mechanisms, however, the interpretation is not straightforward. The electronic effects of the para-substituent in benzylamine also influence the pKa of the amino group and it has been shown by Ramsay and co4328



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workers that this equilibrium can alter the rate of the enzymatic reaction.53 It has also been shown in the same study that the protonated inhibitor is bound to the enzyme. We have shown in a previous report that it only takes 1.9 kcal/mol in terms of free-energy to remove a proton from the protonated dopamine in the active site to the bulk solvent.38 The electrostatic environment influences this value heavily and since the differences in free energies of activation between benzylamine analogues are less than 3 kcal/mol,17,19 a minor change in the electrostatics can contribute significantly to the deprotonation step and thus change the overall energetics of the reaction. However, one must bear in mind that this does not alter the mechanism of the C−H abstraction step. From this viewpoint, one must consider that at a physiological pH of 7.4 the deprotonation of the substrate is, in addition to intrinsic energetics, an integral part of the rate-determining step. Since the reported pKa values of substituted benzylamines in aqueous solution span only 0.8 pKa units54 it is very likely that slightly different electrostatic environments in MAO A and MAO B will change the pKa values of the substrate in the active sites, resulting in changed rate constants. A reliable calculation of the small differences in pKa values would represent a very demanding task for computational studies. As shown by Edmondson and co-workers,54 an alkaline pH of the solution is beneficial for the reaction, since the deprotonation step is made more favorable and possibly even exothermic. The electrostatic environment in both enzymes is, however, not completely identical and could in principle induce different degrees of charge transfer in the reactant complex, as shown by Kästner and co-workers,15 giving rise to a polar nucleophilic mechanism in MAO A and to hydride transfer in MAO B. However, we propose that it is unlikely that such closely related enzymes would employ different mechanistic strategies to accomplish the same task. This would require two different electrostatic preorganizations to stabilize the transition state carbanion in MAO A and a carbocation in MAO B. In our calculation we considered both electronic effects and the effects of thermal fluctuations by considering the total enzyme environment. Steric effects were formally taken into account by the repulsive part of the Lennard-Jones potential and to a minor extent also by electrostatic terms. The enzymes have many degrees of freedom over which a conformational change can be accommodated without a large increase in free energy. This is also one of the reasons why steric strain is not a major factor in catalysis, as has been shown by Warshel and coworkers.55 The pKa is dependent in large part on the electrostatic stabilization (solvation) of the charged species. If the binding modes of various substrates differ substantially as suggested by Edmondson,56 which is unlikely due to the very limited space in the active site, then it would be possible to alter the pKa values. For example, interaction of the tyrosine hydroxyl group with an amino group of the substrate would certainly perturb the pKa values of both hydrogen-bonded partners. Exploring the effect of alternative binding modes on the pKas thus remains a challenge for future studies. A structural comparison of ionizable groups within 16 Å of the flavin N5 atom shown in Figure 1 reveals an almost identical placement of charged groups with an RMSD between centers of charged groups (carboxyl carbon in Asp and Glu, amino nitrogen in Lys, and guanidino carbon in Arg) of only 0.847 Å. The distances from the N5 atom of flavin to the centers of charged groups were also measured and are collected in Table 1, which show that in both MAO A and MAO B these

Table 1. Distances of Centers of Ionizable Groups from the N5 Atom of FADa residue

MAO isoform

distance [Å]

d(MAO A) − d(MAO B) [Å]

Glu446 Glu437 Glu436 Glu427 Glu400 Glu391 Lys395 Lys386 Lys341 Lys332 Asp339 Asp330 Lys305 Lys296 Lys218 Lys209 Glu216 Glu207 Asp64 Asp55 Arg51 Arg42 Arg47 Arg38 Arg45 Arg36

A B A B A B A B A B A B A B A B A B A B A B A B A B

13.3 13.7 12.2 12.6 13.7 14.5 13.9 14.6 15.7 15.9 11.5 11.3 5.6 5.3 8.0 9.3 15.7 15.7 8.6 8.7 9.7 9.9 12.8 13.4 13.7 14.3

−0.4 −0.3 −0.8 −0.7 −0.1 0.2 0.3 −1.3 0.0 −0.2 −0.2 −0.6 −0.5

a

Note the very small differences between the A and B isoforms of MAO. Note that the resolutions for MAO A and MAO B are 2.2 and 1.6 Å, respectively.

are negligible with respect to the uncertainties in the crystal structure of the enzyme. Note that the resolutions for MAO A and MAO B are 2.2 and 1.6 Å, respectively. The remarkable similarity in three-dimensional placement of charged residues extends beyond 16 Å, however, due to the fact that few charged residues do not have a counterpart in the other isoform, a quantitative pairwise RMSD comparison is impossible. The calculated pKa values of MAO A/B pairs Tyr69/60, Lys305/296, Tyr407/398, Tyr444/435, and Tyr197/188 are collected in Table 2. Please note that the results are sensitive to the employed value of the dielectric constant. To approximate the experimental pKa value it is useful to average the results over the dielectric constants 10−16, which in our experience gives the best results.38,51 The superimposed structures of MAO A and MAO B active sites with a docked dopamine molecule and the ionizabile residues in question are shown in Figure 2. Before we start analyzing the pKa results, it is useful to note the fact that experimental aqueous solution pKa values for tyrosine and lysine side chains are assumed to be 10.1 and 10.8, respectively.57 As a consequence, it follows that under physiological conditions tyrosine in aqueous solution is a pretty weak acid and is predominantly present in the neutral Tyr−OH form, whereas the lysine side chain is basic enough to favor protonated monocationic form. Interestingly, in both isoforms, the averaged tyrosine pKa values are increased by 2.8−4.8 pKa units compared to the value in aqueous solution, which clearly demonstrates the hydrophobic nature of the 4329



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Table 2. Calculated pKa Values of Selected Ionizable Residues in the Vicinity of the Active Site of MAO A and MAO Ba residue

MAO isoform

Tyr197 Tyr188 Lys305 Lys296 Tyr407 Tyr398 Tyr444 Tyr435 Tyr69 Tyr60

A B A B A B A B A B

ε = 10 14.51 15.79 11.16 11.12 13.85 14.03 14.68 13.84 13.90 14.06

(0.51) (0.30) (0.29) (0.40) (0.47) (0.50) (0.38) (0.37) (0.39) (0.31)

ε = 11 13.92 15.25 10.47 10.41 13.17 13.28 14.02 13.24 13.26 13.24

(0.43) (0.25) (0.23) (0.31) (0.35) (0.37) (0.32) (0.34) (0.31) (0.25)

ε =12 13.71 14.98 10.63 10.56 12.89 13.00 13.78 13.02 13.06 13.00

ε = 13

(0.39) (0.23) (0.21) (0.29) (0.32) (0.34) (0.30) (0.31) (0.28) (0.23)

13.53 14.75 10.77 10.68 12.65 12.77 13.57 12.83 12.88 12.80

(0.36) (0.21) (0.19) (0.26) (0.30) (0.32) (0.28) (0.29) (0.26) (0.21)

ε = 14 13.38 14.55 10.89 10.79 12.45 12.57 13.39 12.68 12.74 12.63

(0.33) (0.20) (0.18) (0.24) (0.28) (0.29) (0.25) (0.27) (0.25) (0.19)

ε = 16 13.13 14.23 11.08 10.97 12.12 12.25 13.11 12.42 12.49 12.35

(0.29) (0.17) (0.16) (0.21) (0.24) (0.26) (0.22) (0.23) (0.21) (0.17)

average 10−16 13.70 14.93 10.83 10.76 12.86 12.98 13.76 13.01 13.06 13.01

(0.48) (0.55) (0.26) (0.26) (0.61) (0.62) (0.55) (0.50) (0.49) (0.60)

a

Please note that the experimental pKa values in aqueous solution for Tyr is 10.1 and for Lys is 10.8. The values in parentheses represent the standard deviation of pKa values over several conformations.

a higher fluctuation of the pKa value. The pKa value of each amino acid is influenced by the microenvironment provided by the protein structure. The pKa value reflects inter-residue, residue−solvent and long-range electrostatic interactions with other charged residues in the protein or salt ions in solution. The results presented here clearly confirm our hypothesis that the electrostatic environment in MAO A and MAO B is very similar, and it is therefore unlikely that both MAO isoforms catalyze degradation of biogenic amines through two different mechanisms.

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CONCLUDING REMARKS

In this article, we performed a systematic study of the pKa values of titratable groups present in the active site of both A and B isoforms of the monoamine oxidase enzyme and critically compared the nature of both active sites, which were supplemented with dopamine as a typical substrate. Specifically, we have examined the pKas of four tyrosine residues that are part of the so-called aromatic cage and a Lys residue close to the reacting atoms of FAD. The calculations were performed using the full dimensionality of the protein extensively sampled by molecular dynamics simulation. We clearly demonstrated that, for both isozymes, the pKa values for the Tyr and Lys residues in question are not substantially different (1.23 pKa units at maximum), thus providing strong evidence that the electrostatic potential pattern in the active sites of both isozymes is very closely matched. Since enzymes work by preorganized electrostatics, the same electrostatic environment cannot be at the same time suitable for optimally solvating the transition state with a positive and a negative charge build-up, as would be the case in the hydride and polar nucleophilic mechanisms. Superimposition of both experimental X-ray structures also reveals a high similarity in the spatial configuration of charged groups in MAOs. It is therefore unlikely that MAO A and MAO B would work by different chemical mechanisms on the same family of substrates, suggesting that the interesting proposal put forward by Orruet et al.19 is, in this sense, questionable. Further investigation of the catalytic mechanism on the experimental and theoretical QM/MM level in conjunction with different protonation configurations on both isozymes is therefore necessary to elucidate the individual steps in MAO catalysis, which will allow for the development of improved inhibitors with few or no side effects.

Figure 2. Superposition of FAD and ionizable residues surrounding the active site (MAO A is green, MAO B is gray, dopamine molecule in violet).

MAO active sites, in line with the proposal by Edmondson and co-workers.13,27 The increased hydrophobicity also supports the idea that monoamine substrates enter the active site in their unprotonated form, in agreement with our previous study, which revealed that the barrier for the hydride transfer from the protonated substrate is too high for efficient catalysis. All of this is further evidenced in the case of the lysine pKa values, only to a lesser extent. These are unperturbed relative to the aqueous solution, which can be attributed to the position of the investigated lysine residues outside the aromatic cage (Figure 2), where the hydrophobicity is not so pronounced. It is also important to observe that, among isoforms, the average absolute differences over a dielectric constant of 10−16 in the corresponding pKa values are assumed to be 0.05, 0.07, 0.12, 0.75, and 1.23 for MAO A/B pairs Tyr69/60, Lys305/ 296, Tyr407/398, Tyr444/435, and Tyr197/188, respectively. The difference between the Tyr444/435 and Tyr197/188 pairs is more pronounced compared to others, due to the larger impact of the dopamine position on the pKa value, which stems from the interaction between the dopamine and tyrosine hydroxyl groups. Dopamine has two hydroxyl groups on the phenyl ring that are closer to Tyr444/435 and Tyr197/188 than to other investigated residues. Hence, they interact more strongly with the hydroxyl groups of the dopamine resulting in 4330



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(14) Erdem, S. S.; Buyukmenekse, B. Computational Investigation on the Structure-Activity Relationship of the Biradical Mechanism for Monoamine Oxidase. J. Neural Transm. 2011, 118 (7), 1021−1029. (15) Abad, E.; Zenn, R. K.; Kastner, J. Reaction Mechanism of Monoamine Oxidase from QM/MM Calculations. J. Phys. Chem. B 2013, 117, 14238−14246. (16) Vianello, R.; Repič, M.; Mavri, J. How are Biogenic Amines Metabolized by Monoamine Oxidases? Eur. J. Org. Chem. 2012, 2012 (36), 7057−7065. (17) Miller, J. R.; Edmondson, D. E. Structure-Activity Relationships in the Oxidation of Para-substituted Benzylamine Analogues by Recombinant Human Liver Monoamine Oxidase A. Biochemistry 1999, 38 (41), 13670−13683. (18) Walker, M. C.; Edmondson, D. E. Structure-Activity Relationships in the Oxidation of Benzylamine Analogues by Bovine Liver Mitochondrial Monoamine Oxidase B. Biochemistry 1994, 33 (23), 7088−7098. (19) Orru, R.; Aldeco, M.; Edmondson, D. E. Do MAO A and MAO B Utilize the Same Mechanism for the C-H Bond Cleavage Step in Catalysis? Evidence Suggesting Differing Mechanisms. J. Neural Transm. 2013, 120 (6), 847−851. (20) Akyuz, M. A.; Erdem, S. S. Computational Modeling of the Direct Hydride Transfer Mechanism for the MAO Catalyzed Oxidation of Phenethylamine and Benzylamine: ONIOM (QM/ QM) Calculations. J. Neural Transm. 2013, 120 (6), 937−945. (21) Atalay, V. E.; Erdem, S. S. A Comparative Computational Investigation on the Proton and Hydride Transfer Mechanisms of Monoamine Oxidase Using Model Molecules. Comp. Biol. Chem. 2013, 47, 181−191. (22) Warshel, A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. Wiley-Interscience: New York, 1997; p 256. (23) Warshel, A.; Strajbl, M.; Villa, J.; Florian, J. Remarkable Rate Enhancement of Orotidine 5 ′-Monophosphate Decarboxylase is Due to Transition-State Stabilization Rather Than to Ground-State Destabilization. Biochemistry 2000, 39 (48), 14728−14738. (24) Adamczyk, A. J.; Cao, J.; Kamerlin, S. C.; Warshel, A. Catalysis by Dihydrofolate Reductase and Other Enzymes Arises from Electrostatic Preorganization, not Conformational Motions. Proc. Natl. Acad. Sci. U.S.A. 2011, 108 (34), 14115−14120. (25) Warshel, A.; Sharma, P. K.; Kato, M.; Xiang, Y.; Liu, H.; Olsson, M. H. Electrostatic Basis for Enzyme Catalysis. Chem. Rev. 2006, 106 (8), 3210−3235. (26) Warshel, A. Energetics of Enzyme Catalysis. Proc. Natl. Acad. Sci. U.S.A. 1978, 75 (11), 5250−5254. (27) Li, M.; Binda, C.; Mattevi, A.; Edmondson, D. E. Functional Role of the Aromatic Cage in Human Monoamine Oxidase B: Structures and Catalytic Properties of Tyr435 Mutant Proteins. Biochemistry 2006, 45 (15), 4775−4784. (28) Grimsley, G. R.; Scholtz, J. M.; Pace, C. N. A Summary of the Measured pKa Values of the Ionizable Groups in Folded Proteins. Protein Sci. 2009, 18 (1), 247−251. (29) Juffer, A. H. Theoretical Calculations of Acid-Dissociation Constants of Proteins. Biochim. Biol. Cell. 1998, 76 (2−3), 198−209. (30) Milczek, E. M.; Binda, C.; Rovida, S.; Mattevi, A.; Edmondson, D. E. The ‘Gating’ Residues Ile199 and Tyr326 in Human Monoamine Oxidase B Function in Substrate and Inhibitor Recognition. FEBS J. 2011, 278 (24), 4860−4869. (31) Ramsay, R. R. Mechanistic Study of Monoamine Oxidase: Significance for MAO A and MAO B in situ. Vop. Med. Khim. 1997, 43 (6), 457−470. (32) Ramsay, R. R.; Dunford, C.; Gillman, P. K. Methylene Blue and Serotonin Toxicity: Inhibition of Monoamine Oxidase A (MAO A) Confirms a Theoretical Prediction. Br. J. Pharmacol. 2007, 152 (6), 946−951. (33) Ramsay, R. R.; Koerber, S. C.; Singer, T. P. Stopped-Flow Studies on the Mechanism of Oxidation of N-methyl-4-phenyltetrahydropyridine by Bovine Liver Monoamine Oxidase B. Biochemistry 1987, 26 (11), 3045−3050.

ASSOCIATED CONTENT

S Supporting Information *

Detailed computation and modeling information. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(J.M.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.R., M.P., and J.M. would like to thank the Slovenian Research Agency for financial support within the framework of the Program Group P1-0012. R.V. gratefully acknowledges the European Commission for an individual FP7 Marie Curie Career Integration Grant (Contract Number PCIG12-GA2012-334493). Part of this work was supported by COST Action CM1103. We would like to thank Ms. Charlotte C. W. Taft for careful reading of the manuscript and linguistic corrections.

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