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Reaction profiling of the MAO-B catalyzed oxidative deamination of amines in Alzheimer's disease
Journal of Molecular Structure (Theochem) 666–667 (2003) 527–536 www.elsevier.com/locate/theochem
Reaction profiling of the MAO-B catalyzed oxidative deamination of amines in Alzheimer’s disease Donna M. Gasparroa,*, David R.P. Almeidaa, Luca F. Pisterzia, Jason R. Juhasza, Bela Viskolczb, Botond Penkec,d, Imre G. Csizmadiaa,c,e a
Department of Chemistry, University of Toronto, 80 St George St, Toronto, Ont., Canada M5S 3H6 Department of Chemistry, JGyTFK, University of Szeged, P.O. Box 396, H-6701 Szeged, Hungary c Department of Medical Chemistry, University of Szeged, Do´m te´r 8, 6720 Szeged, Hungary d Protein Chemistry Research Group, Hungarian Academy of Sciences, University of Szeged, Do´m te´r 8, 6720 Szeged, Hungary e Global Institute of COmputational Molecular and Materials Science (GIOCOMMS), 1422 Edenrose St, Mississauga, Ont., Canada L5V 1H3 b
Abstract Monoamine oxidase type B (MAO-B) catalyzes the oxidative deamination of various endogenous and exogenous primary, secondary, and tertiary amines. During this reaction, reactive oxygen species (ROS) are produced which contribute to the oxidative stress in a biological system. Further, research has indicated that increased MAO-B levels and increased ROS production may lead to deposition of b-amyloid (Ab) and act as contributing factors to the pathogenesis of Alzheimer’s disease (AD). As such, irreversible MAO-B inhibitors like selegiline, which decrease the rate of MAO-B catalyzed oxidative deamination, and thus the production of ROS, may have therapeutic potential via neuroprotection. In the current study, molecular orbital calculations were performed using the complete basis set 4m (CBS-4m) theoretical framework employed within the GAUSSIAN 98 software to elucidate the full energetic and thermodynamic profile of the MAO-B catalyzed oxidative deamination reaction. Full geometry optimizations were performed on model compounds to reveal the energies ðDEÞ and Gibbs Free Energies ðDGÞ of the oxidative deamination reaction as a means to clarify the details of this paramount reaction and how it relates to AD and selegiline’s therapeutic potential. Results reveal that the oxidative deamination reaction consists of three successive energy-consuming steps for all amines. Trends indicate that for all amines, as amine substitution increases: (1) energetic investments needed for steps 1 and 3 increase, (2) DEReaction and DGReaction values for the oxidative deamination reaction as a whole increase, (3) differences between DGReaction and DEReaction values increase, and (4) DG and DE values for step 2 of the reaction decrease. The second step of the MAO-B catalyzed reaction is the only step, where increased amine substitution correlates with decreased DE and DG values. Due to the fact that step two possesses the lowest investment of energy for tertiary and secondary amines but the highest investment of energy for primary amines, it can be postulated that MAO-B has preferred secondary and tertiary substrates. The fact that step 2 is the rate-limiting step relates to the notion that it modulates the potential to increase oxidative stress, and thus, must be tightly regulated to maintain oxidant and anti-oxidant homeostasis. Finally, in disease states such as AD, where increased MAO-B levels have been documented, it is possible that
* Corresponding author. E-mail addresses: [email protected] (D.M. Gasparro), [email protected] (D.R.P. Almeida), [email protected] (L.F. Pisterzi), [email protected] (J.R. Juhasz), [email protected] (B. Viskolcz), [email protected] (B. Penke), [email protected] (I.G. Csizmadia). 0166-1280/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.theochem.2003.08.077
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MAO-B mediated oxidative stress is a contributing factor to AD. Thus, MAO-B inhibition by drugs like selegiline will provide neuroprotective benefits and help to prevent AD pathogenesis. q 2003 Elsevier B.V. All rights reserved. Keywords: Monoamine oxidase; Monoamine oxidase type B; Oxidative deamination; Reactive oxygen species; Alzheimer’s disease; Complete basis set 4m
1. Introduction Monoamine oxidase type B (MAO-B) is an integral membrane protein in outer mitochondrial membranes and is found in neuronal and non-neuronal cells in the brain and in peripheral organs [1]. MAO-B is a constitutive enzyme found predominantly in serotonergic neurons of the brain as well as in glial cells and at high concentrations in the thalamus, stratium, cortex and the brainstem [2,3]. MAO-B preferentially breaks down b-phenylethylamine and is selectively and irreversibly inhibited by the drug selegiline (also known as deprenyl) [4]. The half-life of MAO-B is approximately 30 days in the CNS; thus, once selegiline binds to MAO-B, the enzyme remains inactive until it is degraded and new proteins are synthesized [5]. MAO-B exists as a protein homodimer with the Cterminal cysteine of each monomer covalently bound to the 8a position of a flavin adenine dinucleotide (FAD) [6]. As a flavoenzyme, MAO-B catalyzes the oxidative deamination of various neurotransmitters (dopamine, serotonin, noradrenaline) and amines (select primary, secondary, and tertiary amines) [7, 8]. MAO-B is responsible for the oxidative deamination of various amines to an imine followed by the formation of an aldehyde or ketone and a corresponding amine (Fig. 1) [5]. The rate of oxidation of a particular amine by MAO-B is dependent on amine concentration, availability of oxygen (which oxidizes chemically reduced MAO-B via the oxidation of FADH2 to FAD), and the concentration of MAO-B in the mitochondrial outer membranes [3]. MAO-B inhibitors such as selegiline decrease the availability of MAO-B and thus, the rate of oxidative deamination [3]. There are six postulated modes of action for selegiline (1) induces anti-apoptotic activity, (2) increases the amount of N-acetylated polyamines hence acting as an antagonist at NMDA receptors, (3) increases nitric oxide synthesis, (4) increases monoamine levels, (5) induces anti-oxidant
enzymes such as superoxide dismutase and catalase, and (6) inhibits MAO-B [9]. It is essential to note that the last two modes of action mentioned are antioxidant activities of selegiline. The proposed molecular mechanism for MAO-B catalysis involves the formation of hydrogen peroxide (H2O2) concomitant with FADH2 oxidation to FAD (Fig. 1) [10]. If H2O2 is not detoxified by glutathione peroxidase, hydroxyl radicals are generated via the Fenton reaction which may propagate free radical damage (Fig. 1) [11]. The hydroxyl radical is cytotoxic due to its high reactivity towards lipids, proteins, and DNA [12,13]. Anti-oxidant enzymes such as catalase and superoxide dismutase are responsible for scavenging radicals, however, these enzymes are found at lowest concentrations in the brain relative to the rest of the body. The formation of free radicals via MAO-B oxidation is implicated in the neurodegeneration associated with Alzheimer’s disease (AD) and in the pathophysiology of aging [14 –16]. As levels of MAO-B increase, the brain dopamine system has a corresponding increase in vulnerability to damage via oxidative stress [1]. Various data implicates MAO-B oxidation in AD: postmortem AD patients show an increase in MAO-B levels, MAO-B is expressed in astrocytes of senile plaques in AD patients, and increased binding of radiolabelled selegiline to MAO-B in AD patients [1]. Given the above, selegiline can be viewed as a neuroprotective agent able to delay the progression of neuronal cell death vis-a`-vis anti-oxidant modes of action—especially in its inhibition of MAO-B. Consequently, selegiline is currently undergoing human trials to test its efficacy in alleviating AD pathology [13]. Selegiline is also used to treat other neurological diseases such as Parkinson’s disease, global ischemia, narcolepsy, and Gilles de la Tourette Syndrome [15,17]. Moreover, since the MAO-B catalyzed oxidative deamination reaction is pathologically linked to AD
D.M. Gasparro et al. / Journal of Molecular Structure (Theochem) 666–667 (2003) 527–536
Fig. 1. General reaction path of the MAO-B catalyzed oxidative deamination of primary, secondary, and tertiary amines. Imine formation (step 2) occurs concomitantly with reactive oxygen species (ROS) production, which may contribute to oxidative stress and the neurodegeneration in disease stated like Alzheimer’s disease. The irreversible MAO-B inhibitor, selegiline, blocks the oxidative deamination reaction.
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and MAO-B inhibition by selegiline is a fundamental basis of selegiline’s efficacy towards improving neurological disorders, full characterization of the MAO-B reaction is critical to understanding its role in these disease pathologies. In the current study, the oxidative deamination reaction (presented in Fig. 1) is thermodynamically quantified to elucidate any molecular details or characteristics that may be relevant to AD and selegiline’s mode of action in improving neurodegenerative disorders such as AD. 2. Methods The current study focuses on the thermodynamic assessment and characterization of the MAO-B catalyzed oxidative deamination of primary, secondary, and tertiary amines. The reaction coordinate possesses three distinct steps (and four corresponding reaction plateaus): (1) deprotonation of a protonated amine (primary, secondary, or tertiary) to yield a neutral amine, (2) oxidation of a neutral amine to an imine intermediate, and (3) hydration of an imine to an aldehyde or ketone and a corresponding amine (Fig. 1). In this study, FAD and FADH2 are modeled with Liþ and LiH, respectively; and the process of FAD oxidation to FADH2 is computed as a one-step hydride reduction of Liþ to LiH (Fig. 1).
To characterize the full oxidative deamination reaction, model species involved in the reaction coordinate (Table 1) were investigated with molecular orbital (MO) calculations using the GAUSSIAN 98 software [18]. Molecular structure, stereochemistry, and geometry of all species were exclusively defined in terms of GAUSSIAN 98 internal Cartesian coordinates. All calculations were performed at the complete basis set 4m (CBS-4m; m refers to the use of Minimal Population localization) [19] level of theory in the gas phase ð1 ¼ 0:0Þ to extract the best possible energies for this process. Initially, all species involved in this reaction were subject to full geometry optimizations and their total energy ðEÞ and Gibbs Free Energy ðGÞ calculated (Table 1). Subsequently, E and G values were summated for each respective step of the reaction path according to the molecular species present at that step, while ensuring proper stoichiometry and charge conservation throughout the reaction coordinate. These latter E and G values were used to calculate the energy of reaction ðDEÞ and Gibbs Free Energy of reaction ðDGÞ between all steps of the reaction path. Finally, energy and Gibbs Free energy of reaction for the complete oxidative deamination reaction (denoted as DEReaction and DGReaction ) were calculated. All graphical data were plotted using Axum 5.0 [20].
Table 1 Model molecules involved in the MAO-B catalyzed oxidative deamination of primary, secondary and tertiary amines Chemical name
Chemical formula
Energy (SCF) (kcal/mol)
Free energy, G (kcal/mol)
Primary amine Protonated primary amine Secondary amine Protonated secondary amine Tertiary amine Protonated tertiary amine Lithium Lithium hydride Water Hydronium Imine Methyl imine Dimethyl imine Formaldehyde Non-substituted amine (ammonia)
[NH3CH3] [NH4CH3]þ [NH(CH3)2] [NH2(CH3)2]þ [N(CH3)3] [NH(CH3)3]þ [Li]þ [LiH] [H2O] [H3O]þ [CH2NH2]þ [CH2NH(CH3)]þ [CH2N(CH3)2]þ [H2CO] [NH3]
295.246615 295.608019 2134.282784 2134.656043 2173.320861 2173.701913 27.235840 27.985592 276.053394 276.330925 294.417284 2133.469364 2172.518679 2113.907358 256.214201
295.719235 296.060638 2134.950356 2135.302490 2174.186066 2174.545041 27.248587 28.041151 276.367546 276.625856 294.835524 2134.083487 2173.330591 2114.381593 256.493922
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Fig. 2. Thermodynamic and energetic profiles of the MAO-B catalyzed oxidative deamination of primary amines calculated at the CBS-4m level of theory.
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3. Results and discussion Thermodynamic and energetic data revealed that the oxidative deamination reaction catalyzed by MAO-B consists of three successive energy-consuming steps for primary (Fig. 2), secondary (Fig. 3), and tertiary (Fig. 4) amines; all amines computed in this study revealed this same profile. A general decreasing trend in DEReaction and DGReaction is evident from tertiary amine (DGReaction ¼ 170:34 kcal/mol, DEReaction ¼ 163:37 kcal/mol), to secondary amine (DGReaction ¼ 163:16 kcal/mol, DEReaction ¼ 157:29 kcal/mol), to primary amine (DGReaction ¼ 152:79 kcal/mol, DEReaction ¼ 147:48 kcal/mol). As amine substitution increases, so does the energetic investment of the oxidative deamination reaction (Eqs. (1) and (2) and Figs. 2 –4)
ð1Þ
DEReaction ð38 amineÞ . DEReaction ð28 amineÞ . DEReaction ð18 amineÞ
DGsteps 1;3 ð38 amineÞ . DGsteps 1;3 ð28 amineÞ . DGsteps 1;3 ð18 amineÞ
ð3Þ
DEsteps 1;3 ð38 amineÞ . DEsteps 1;3 ð28 amineÞ . DEsteps 1;3 ð18 amineÞ
ð4Þ
DGstep 2 ð18 amineÞ . DGstep 2 ð28 amineÞ . DGstep 2 ð38 amineÞ
ð5Þ
DEstep 2 ð18 amineÞ . DEstep 2 ð28 amineÞ
DGReaction ð38 amineÞ . DGReaction ð28 amineÞ . DGReaction ð18 amineÞ
species (ROS) formation; here, as substitution increases, DG and DE values decrease (Eqs. (5) and (6) and Figs. 2 –4). The second step of the MAO-B catalyzed reaction is the only step of the reaction that not only deviates, but also is opposite to the trend indicated in Eqs. (1) –(4)
ð2Þ
Moreover, as the substitution of an amine increases, the difference between DGReaction and DEReaction for the oxidative deamination of that amine also increases (Fig. 5). Given that DG values indicate the amount of work-energy, while DE values indicate the total energy of a system, along with the fact that as MAO-B substrates get more complex, the substrates DG and DE values deviate further from each other, i can be predicted that DG values will be the best descriptive terms for the thermodynamics of the oxidative deamination reaction. Thus, for the purpose of this study, the calculated DG values will be deemed as the best prognosticators for MAO-B catalyzed oxidative deamination reaction. As stated above, as substitution increases, so does the DGReaction and DEReaction values. This same trend is present for the DG and DE values for steps 1 and 3 of the oxidative deamination reaction (Eqs. (3) and (4) and Figs. 2– 4). Overall, the only exception to this trend of increasing energy consumption with increasing amine substitution occurs in the second step of the reaction, where oxidation of a neutral amine to an imine intermediate is coupled to reactive oxygen
. DEstep 2 ð38 amineÞ
ð6Þ
Thermodynamically, step two possesses the lowest investment of energy of the entire reaction for tertiary and secondary amines, but possesses the highest investment of energy for primary amines. Thus MAO-B may preferentially bind primary amine substrates (such as b-phenylethylamine), however, once a primary amine is bound to MAO-B a high energetic barrier for the second step is encountered, which is uncharacteristic of secondary and tertiary amines. It can then be postulated that MAO-B has the highest energetic barrier at the second step for primary amines as compaired to secondary and tertiary amines, as a means to control ROS formation. This is supported by the fact that both neurotransmitters (which are often primary amines such as dopamine and serotonin), and mitochondria, which contain MAO-B, are abundant in neurons. Thus the high energetic barrier of step two of the MAO-B reaction may serve to decrease ROS formation that would otherwise be excessive since both the substrate (primary amine neurotransmitters) and the enzyme (MAO-B) are in abundance in neurons. According to kinetic studies, the rate-limiting step in the MAO-B catalyzed reaction is the oxidative cleavage of the C –H bond that is alpha to the amino group (step 2) [1]. Having step 2 as the rate-limiting step is consistent with the idea that this step is
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Fig. 3. Thermodynamic and energetic profiles of the MAO-B catalyzed oxidative deamination of secondary amines calculated at the CBS-4m level of theory.
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Fig. 4. Thermodynamic and energetic profiles of the MAO-B catalyzed oxidative deamination of tertiary amines calculated at the CBS-4m level of theory.
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Fig. 5. Graphical representation of the increasing disparity between CBS-4m calculated DG and DE values as amine substitution increases.
deleterious to living cells because it increases oxidative stress; therefore, it is imperative that this step be kept at a carefully defined and controlled rate. This is to say, the ROS forming step is rate-limiting in order to protect the cell from oxidative stress and to maintain oxidant and anti-oxidant homeostasis.
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AD patients [1]) will lead to oxidative abnormalities. Hence, the therapeutic inhibition of this reaction by a pharmaceutical agent like selegiline will avoid oxidative abnormalities in the cell and subsequently prevent b-amyloid (Ab) deposition (Fig. 6) [21]. Indirectly, oxidative abnormalities promote calcium abnormalities which promote Ab formation (Fig. 6) [21]. Since Ab is a defining feature of AD, and given Ab deposition is promoted by ROS, it can be inferred that the inhibition of the rate-limiting step (step 2) of the MAO-B catalyzed reaction is neuroprotective via an anti-oxidant mode of action and may help prevent AD pathogenesis.
Acknowledgements One of the authors (IGC) wishes to thank the Ministry of Education for a Szent-Gyo¨rgyi Visiting Professorship.
References 4. Conclusion It can be postulated that in disease states such as AD, increased MAO-B levels (as seen in post mortem
Fig. 6. The MAO-B oxidative deamination reaction is controlled by having the reactive oxygen species forming step as the rate-limiting step. However, it is postulated that increased MAO-B promotes increased ROS production, oxidative and calcium abnormalities, and deposition of b-amyloid (Ab), which may lead to Alzheimer’s disease. Consequently, MAO-B inhibition by selegiline is a neuroprotective reaction via an anti-oxidant mode of action.
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Reaction profiling of the MAO-B catalyzed oxidative deamination of amines in Alzheimer’s disease Donna M. Gasparroa,*, David R.P. Almeidaa, Luca F. Pisterzia, Jason R. Juhasza, Bela Viskolczb, Botond Penkec,d, Imre G. Csizmadiaa,c,e a
Department of Chemistry, University of Toronto, 80 St George St, Toronto, Ont., Canada M5S 3H6 Department of Chemistry, JGyTFK, University of Szeged, P.O. Box 396, H-6701 Szeged, Hungary c Department of Medical Chemistry, University of Szeged, Do´m te´r 8, 6720 Szeged, Hungary d Protein Chemistry Research Group, Hungarian Academy of Sciences, University of Szeged, Do´m te´r 8, 6720 Szeged, Hungary e Global Institute of COmputational Molecular and Materials Science (GIOCOMMS), 1422 Edenrose St, Mississauga, Ont., Canada L5V 1H3 b
Abstract Monoamine oxidase type B (MAO-B) catalyzes the oxidative deamination of various endogenous and exogenous primary, secondary, and tertiary amines. During this reaction, reactive oxygen species (ROS) are produced which contribute to the oxidative stress in a biological system. Further, research has indicated that increased MAO-B levels and increased ROS production may lead to deposition of b-amyloid (Ab) and act as contributing factors to the pathogenesis of Alzheimer’s disease (AD). As such, irreversible MAO-B inhibitors like selegiline, which decrease the rate of MAO-B catalyzed oxidative deamination, and thus the production of ROS, may have therapeutic potential via neuroprotection. In the current study, molecular orbital calculations were performed using the complete basis set 4m (CBS-4m) theoretical framework employed within the GAUSSIAN 98 software to elucidate the full energetic and thermodynamic profile of the MAO-B catalyzed oxidative deamination reaction. Full geometry optimizations were performed on model compounds to reveal the energies ðDEÞ and Gibbs Free Energies ðDGÞ of the oxidative deamination reaction as a means to clarify the details of this paramount reaction and how it relates to AD and selegiline’s therapeutic potential. Results reveal that the oxidative deamination reaction consists of three successive energy-consuming steps for all amines. Trends indicate that for all amines, as amine substitution increases: (1) energetic investments needed for steps 1 and 3 increase, (2) DEReaction and DGReaction values for the oxidative deamination reaction as a whole increase, (3) differences between DGReaction and DEReaction values increase, and (4) DG and DE values for step 2 of the reaction decrease. The second step of the MAO-B catalyzed reaction is the only step, where increased amine substitution correlates with decreased DE and DG values. Due to the fact that step two possesses the lowest investment of energy for tertiary and secondary amines but the highest investment of energy for primary amines, it can be postulated that MAO-B has preferred secondary and tertiary substrates. The fact that step 2 is the rate-limiting step relates to the notion that it modulates the potential to increase oxidative stress, and thus, must be tightly regulated to maintain oxidant and anti-oxidant homeostasis. Finally, in disease states such as AD, where increased MAO-B levels have been documented, it is possible that
* Corresponding author. E-mail addresses: [email protected] (D.M. Gasparro), [email protected] (D.R.P. Almeida), [email protected] (L.F. Pisterzi), [email protected] (J.R. Juhasz), [email protected] (B. Viskolcz), [email protected] (B. Penke), [email protected] (I.G. Csizmadia). 0166-1280/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.theochem.2003.08.077
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MAO-B mediated oxidative stress is a contributing factor to AD. Thus, MAO-B inhibition by drugs like selegiline will provide neuroprotective benefits and help to prevent AD pathogenesis. q 2003 Elsevier B.V. All rights reserved. Keywords: Monoamine oxidase; Monoamine oxidase type B; Oxidative deamination; Reactive oxygen species; Alzheimer’s disease; Complete basis set 4m
1. Introduction Monoamine oxidase type B (MAO-B) is an integral membrane protein in outer mitochondrial membranes and is found in neuronal and non-neuronal cells in the brain and in peripheral organs [1]. MAO-B is a constitutive enzyme found predominantly in serotonergic neurons of the brain as well as in glial cells and at high concentrations in the thalamus, stratium, cortex and the brainstem [2,3]. MAO-B preferentially breaks down b-phenylethylamine and is selectively and irreversibly inhibited by the drug selegiline (also known as deprenyl) [4]. The half-life of MAO-B is approximately 30 days in the CNS; thus, once selegiline binds to MAO-B, the enzyme remains inactive until it is degraded and new proteins are synthesized [5]. MAO-B exists as a protein homodimer with the Cterminal cysteine of each monomer covalently bound to the 8a position of a flavin adenine dinucleotide (FAD) [6]. As a flavoenzyme, MAO-B catalyzes the oxidative deamination of various neurotransmitters (dopamine, serotonin, noradrenaline) and amines (select primary, secondary, and tertiary amines) [7, 8]. MAO-B is responsible for the oxidative deamination of various amines to an imine followed by the formation of an aldehyde or ketone and a corresponding amine (Fig. 1) [5]. The rate of oxidation of a particular amine by MAO-B is dependent on amine concentration, availability of oxygen (which oxidizes chemically reduced MAO-B via the oxidation of FADH2 to FAD), and the concentration of MAO-B in the mitochondrial outer membranes [3]. MAO-B inhibitors such as selegiline decrease the availability of MAO-B and thus, the rate of oxidative deamination [3]. There are six postulated modes of action for selegiline (1) induces anti-apoptotic activity, (2) increases the amount of N-acetylated polyamines hence acting as an antagonist at NMDA receptors, (3) increases nitric oxide synthesis, (4) increases monoamine levels, (5) induces anti-oxidant
enzymes such as superoxide dismutase and catalase, and (6) inhibits MAO-B [9]. It is essential to note that the last two modes of action mentioned are antioxidant activities of selegiline. The proposed molecular mechanism for MAO-B catalysis involves the formation of hydrogen peroxide (H2O2) concomitant with FADH2 oxidation to FAD (Fig. 1) [10]. If H2O2 is not detoxified by glutathione peroxidase, hydroxyl radicals are generated via the Fenton reaction which may propagate free radical damage (Fig. 1) [11]. The hydroxyl radical is cytotoxic due to its high reactivity towards lipids, proteins, and DNA [12,13]. Anti-oxidant enzymes such as catalase and superoxide dismutase are responsible for scavenging radicals, however, these enzymes are found at lowest concentrations in the brain relative to the rest of the body. The formation of free radicals via MAO-B oxidation is implicated in the neurodegeneration associated with Alzheimer’s disease (AD) and in the pathophysiology of aging [14 –16]. As levels of MAO-B increase, the brain dopamine system has a corresponding increase in vulnerability to damage via oxidative stress [1]. Various data implicates MAO-B oxidation in AD: postmortem AD patients show an increase in MAO-B levels, MAO-B is expressed in astrocytes of senile plaques in AD patients, and increased binding of radiolabelled selegiline to MAO-B in AD patients [1]. Given the above, selegiline can be viewed as a neuroprotective agent able to delay the progression of neuronal cell death vis-a`-vis anti-oxidant modes of action—especially in its inhibition of MAO-B. Consequently, selegiline is currently undergoing human trials to test its efficacy in alleviating AD pathology [13]. Selegiline is also used to treat other neurological diseases such as Parkinson’s disease, global ischemia, narcolepsy, and Gilles de la Tourette Syndrome [15,17]. Moreover, since the MAO-B catalyzed oxidative deamination reaction is pathologically linked to AD
D.M. Gasparro et al. / Journal of Molecular Structure (Theochem) 666–667 (2003) 527–536
Fig. 1. General reaction path of the MAO-B catalyzed oxidative deamination of primary, secondary, and tertiary amines. Imine formation (step 2) occurs concomitantly with reactive oxygen species (ROS) production, which may contribute to oxidative stress and the neurodegeneration in disease stated like Alzheimer’s disease. The irreversible MAO-B inhibitor, selegiline, blocks the oxidative deamination reaction.
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and MAO-B inhibition by selegiline is a fundamental basis of selegiline’s efficacy towards improving neurological disorders, full characterization of the MAO-B reaction is critical to understanding its role in these disease pathologies. In the current study, the oxidative deamination reaction (presented in Fig. 1) is thermodynamically quantified to elucidate any molecular details or characteristics that may be relevant to AD and selegiline’s mode of action in improving neurodegenerative disorders such as AD. 2. Methods The current study focuses on the thermodynamic assessment and characterization of the MAO-B catalyzed oxidative deamination of primary, secondary, and tertiary amines. The reaction coordinate possesses three distinct steps (and four corresponding reaction plateaus): (1) deprotonation of a protonated amine (primary, secondary, or tertiary) to yield a neutral amine, (2) oxidation of a neutral amine to an imine intermediate, and (3) hydration of an imine to an aldehyde or ketone and a corresponding amine (Fig. 1). In this study, FAD and FADH2 are modeled with Liþ and LiH, respectively; and the process of FAD oxidation to FADH2 is computed as a one-step hydride reduction of Liþ to LiH (Fig. 1).
To characterize the full oxidative deamination reaction, model species involved in the reaction coordinate (Table 1) were investigated with molecular orbital (MO) calculations using the GAUSSIAN 98 software [18]. Molecular structure, stereochemistry, and geometry of all species were exclusively defined in terms of GAUSSIAN 98 internal Cartesian coordinates. All calculations were performed at the complete basis set 4m (CBS-4m; m refers to the use of Minimal Population localization) [19] level of theory in the gas phase ð1 ¼ 0:0Þ to extract the best possible energies for this process. Initially, all species involved in this reaction were subject to full geometry optimizations and their total energy ðEÞ and Gibbs Free Energy ðGÞ calculated (Table 1). Subsequently, E and G values were summated for each respective step of the reaction path according to the molecular species present at that step, while ensuring proper stoichiometry and charge conservation throughout the reaction coordinate. These latter E and G values were used to calculate the energy of reaction ðDEÞ and Gibbs Free Energy of reaction ðDGÞ between all steps of the reaction path. Finally, energy and Gibbs Free energy of reaction for the complete oxidative deamination reaction (denoted as DEReaction and DGReaction ) were calculated. All graphical data were plotted using Axum 5.0 [20].
Table 1 Model molecules involved in the MAO-B catalyzed oxidative deamination of primary, secondary and tertiary amines Chemical name
Chemical formula
Energy (SCF) (kcal/mol)
Free energy, G (kcal/mol)
Primary amine Protonated primary amine Secondary amine Protonated secondary amine Tertiary amine Protonated tertiary amine Lithium Lithium hydride Water Hydronium Imine Methyl imine Dimethyl imine Formaldehyde Non-substituted amine (ammonia)
[NH3CH3] [NH4CH3]þ [NH(CH3)2] [NH2(CH3)2]þ [N(CH3)3] [NH(CH3)3]þ [Li]þ [LiH] [H2O] [H3O]þ [CH2NH2]þ [CH2NH(CH3)]þ [CH2N(CH3)2]þ [H2CO] [NH3]
295.246615 295.608019 2134.282784 2134.656043 2173.320861 2173.701913 27.235840 27.985592 276.053394 276.330925 294.417284 2133.469364 2172.518679 2113.907358 256.214201
295.719235 296.060638 2134.950356 2135.302490 2174.186066 2174.545041 27.248587 28.041151 276.367546 276.625856 294.835524 2134.083487 2173.330591 2114.381593 256.493922
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Fig. 2. Thermodynamic and energetic profiles of the MAO-B catalyzed oxidative deamination of primary amines calculated at the CBS-4m level of theory.
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3. Results and discussion Thermodynamic and energetic data revealed that the oxidative deamination reaction catalyzed by MAO-B consists of three successive energy-consuming steps for primary (Fig. 2), secondary (Fig. 3), and tertiary (Fig. 4) amines; all amines computed in this study revealed this same profile. A general decreasing trend in DEReaction and DGReaction is evident from tertiary amine (DGReaction ¼ 170:34 kcal/mol, DEReaction ¼ 163:37 kcal/mol), to secondary amine (DGReaction ¼ 163:16 kcal/mol, DEReaction ¼ 157:29 kcal/mol), to primary amine (DGReaction ¼ 152:79 kcal/mol, DEReaction ¼ 147:48 kcal/mol). As amine substitution increases, so does the energetic investment of the oxidative deamination reaction (Eqs. (1) and (2) and Figs. 2 –4)
ð1Þ
DEReaction ð38 amineÞ . DEReaction ð28 amineÞ . DEReaction ð18 amineÞ
DGsteps 1;3 ð38 amineÞ . DGsteps 1;3 ð28 amineÞ . DGsteps 1;3 ð18 amineÞ
ð3Þ
DEsteps 1;3 ð38 amineÞ . DEsteps 1;3 ð28 amineÞ . DEsteps 1;3 ð18 amineÞ
ð4Þ
DGstep 2 ð18 amineÞ . DGstep 2 ð28 amineÞ . DGstep 2 ð38 amineÞ
ð5Þ
DEstep 2 ð18 amineÞ . DEstep 2 ð28 amineÞ
DGReaction ð38 amineÞ . DGReaction ð28 amineÞ . DGReaction ð18 amineÞ
species (ROS) formation; here, as substitution increases, DG and DE values decrease (Eqs. (5) and (6) and Figs. 2 –4). The second step of the MAO-B catalyzed reaction is the only step of the reaction that not only deviates, but also is opposite to the trend indicated in Eqs. (1) –(4)
ð2Þ
Moreover, as the substitution of an amine increases, the difference between DGReaction and DEReaction for the oxidative deamination of that amine also increases (Fig. 5). Given that DG values indicate the amount of work-energy, while DE values indicate the total energy of a system, along with the fact that as MAO-B substrates get more complex, the substrates DG and DE values deviate further from each other, i can be predicted that DG values will be the best descriptive terms for the thermodynamics of the oxidative deamination reaction. Thus, for the purpose of this study, the calculated DG values will be deemed as the best prognosticators for MAO-B catalyzed oxidative deamination reaction. As stated above, as substitution increases, so does the DGReaction and DEReaction values. This same trend is present for the DG and DE values for steps 1 and 3 of the oxidative deamination reaction (Eqs. (3) and (4) and Figs. 2– 4). Overall, the only exception to this trend of increasing energy consumption with increasing amine substitution occurs in the second step of the reaction, where oxidation of a neutral amine to an imine intermediate is coupled to reactive oxygen
. DEstep 2 ð38 amineÞ
ð6Þ
Thermodynamically, step two possesses the lowest investment of energy of the entire reaction for tertiary and secondary amines, but possesses the highest investment of energy for primary amines. Thus MAO-B may preferentially bind primary amine substrates (such as b-phenylethylamine), however, once a primary amine is bound to MAO-B a high energetic barrier for the second step is encountered, which is uncharacteristic of secondary and tertiary amines. It can then be postulated that MAO-B has the highest energetic barrier at the second step for primary amines as compaired to secondary and tertiary amines, as a means to control ROS formation. This is supported by the fact that both neurotransmitters (which are often primary amines such as dopamine and serotonin), and mitochondria, which contain MAO-B, are abundant in neurons. Thus the high energetic barrier of step two of the MAO-B reaction may serve to decrease ROS formation that would otherwise be excessive since both the substrate (primary amine neurotransmitters) and the enzyme (MAO-B) are in abundance in neurons. According to kinetic studies, the rate-limiting step in the MAO-B catalyzed reaction is the oxidative cleavage of the C –H bond that is alpha to the amino group (step 2) [1]. Having step 2 as the rate-limiting step is consistent with the idea that this step is
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Fig. 3. Thermodynamic and energetic profiles of the MAO-B catalyzed oxidative deamination of secondary amines calculated at the CBS-4m level of theory.
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Fig. 4. Thermodynamic and energetic profiles of the MAO-B catalyzed oxidative deamination of tertiary amines calculated at the CBS-4m level of theory.
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Fig. 5. Graphical representation of the increasing disparity between CBS-4m calculated DG and DE values as amine substitution increases.
deleterious to living cells because it increases oxidative stress; therefore, it is imperative that this step be kept at a carefully defined and controlled rate. This is to say, the ROS forming step is rate-limiting in order to protect the cell from oxidative stress and to maintain oxidant and anti-oxidant homeostasis.
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AD patients [1]) will lead to oxidative abnormalities. Hence, the therapeutic inhibition of this reaction by a pharmaceutical agent like selegiline will avoid oxidative abnormalities in the cell and subsequently prevent b-amyloid (Ab) deposition (Fig. 6) [21]. Indirectly, oxidative abnormalities promote calcium abnormalities which promote Ab formation (Fig. 6) [21]. Since Ab is a defining feature of AD, and given Ab deposition is promoted by ROS, it can be inferred that the inhibition of the rate-limiting step (step 2) of the MAO-B catalyzed reaction is neuroprotective via an anti-oxidant mode of action and may help prevent AD pathogenesis.
Acknowledgements One of the authors (IGC) wishes to thank the Ministry of Education for a Szent-Gyo¨rgyi Visiting Professorship.
References 4. Conclusion It can be postulated that in disease states such as AD, increased MAO-B levels (as seen in post mortem
Fig. 6. The MAO-B oxidative deamination reaction is controlled by having the reactive oxygen species forming step as the rate-limiting step. However, it is postulated that increased MAO-B promotes increased ROS production, oxidative and calcium abnormalities, and deposition of b-amyloid (Ab), which may lead to Alzheimer’s disease. Consequently, MAO-B inhibition by selegiline is a neuroprotective reaction via an anti-oxidant mode of action.
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