Rat MAO Research Articles

2H Kinetic Isotope Effects and pH Dependence of Catalysis as Mechanistic Probes of Rat Monoamine Oxidase A: Comparisons with the Human Enzyme

Monoamine oxidase A (MAO A) is a mitochondrial outer membrane-bound flavoenzyme important in the regulation of serotonin and dopamine levels. Because the rat is extensively used as an animal model in drug studies, it is important to understand how rat MAO A behaves in comparison with the more extensively studied human enzyme. For many reversible inhibitors, rat MAO A exhibits K(i) values similar to those of human MAO A. The pH profile of k(cat) for rat MAO A shows a pK(a) of 8.2 ± 0.1 for the benzylamine ES complex and pK(a) values of 7.5 ± 0.1 and 7.6 ± 0.1 for the ES complexes with p-CF(3)-(1)H- and p-CF(3)-(2)H-benzylamine, respectively. In contrast to the human enzyme, the rat enzyme exhibits a single pK(a) value (8.3 ± 0.1) with k(cat)/K(m) for benzylamine versus pH and pK(a) values of 7.8 ± 0.1 and 8.1 ± 0.2 for the ascending limbs, respectively, of k(cat)/K(m) versus pH profiles for p-CF(3)-(1)H- and p-CF(3)-(2)H-benzylamine and 9.3 ± 0.1 and 9.1 ± 0.2 for the descending limbs, respectively. The oxidation of para-substituted benzylamine substrate analogues by rat MAO A has large deuterium kinetic isotope effects on k(cat) and on k(cat)/K(m). These effects are pH-independent and range from 7 to 14, demonstrating a rate-limiting α-C-H bond cleavage step in catalysis. Quantitative structure-activity correlations of log k(cat) with the electronic substituent parameter (σ) at pH 7.5 and 9.0 show a dominant contribution with positive ρ values (1.2-1.3) and a pH-independent negative contribution from the steric term. Quantitative structure-activity relationship analysis of the binding affinities of the para-substituted benzylamine analogues for rat MAO A shows an increased van der Waals volume (V(w)) increases the affinity of the deprotonated amine for the enzyme. These results demonstrate that rat MAO A exhibits functional properties similar but not identical with those of the human enzyme and provide additional support for C-H bond cleavage via a polar nucleophilic mechanism.

Topological Probes of Monoamine Oxidases A and B in Rat Liver Mitochondria: Inhibition by TEMPO-Substituted Pargyline Analogues and Inactivation by Proteolysis

TEMPO-substituted pargyline analogues differentially inhibit recombinant human monoamine oxidase A (MAO A) and B (MAO B) in intact yeast mitochondria, suggesting these membrane-bound enzymes are located on differing faces of the mitochondrial outer membrane [Upadhyay, A., and Edmondson, D. E. (2009) Biochemistry 48, 3928]. This approach is extended to the recombinant rat enzymes and to rat liver mitochondria. The differential specificities exhibited for human MAO A and MAO B by the m- and p-amido TEMPO pargylines are not as absolute with the rat enzymes. Similar patterns of reactivity are observed for rat MAO A and B in mitochondrial outer membrane preparations expressed in Pichia pastoris or isolated from rat liver. In intact yeast mitochondria, recombinant rat MAO B is inhibited by the pargyline analogue whereas MAO A activity shows no inhibition. Intact rat liver mitochondria exhibit an inhibition pattern opposite to that observed in yeast where MAO A is inhibited and MAO B activity is unaffected. Protease inactivation studies show specificity in that MAO A is sensitive to trypsin whereas MAO B is sensitive to β-chymotrypsin. In intact mitochondrial preparations, MAO A is readily inactivated in rat liver but not in yeast upon trypsin treatment and MAO B is readily inactivated by β-chymotrypsin in yeast but not in rat liver. These data show MAO A is oriented on the cytosolic face and MAO B is situated on the surface facing the intermembrane space of the mitochondrial outer membrane in rat liver. The differential mitochondrial outer membrane topology of MAO A and MAO B is relevant to their inhibition by drugs designed to be cardioprotectants or neuroprotectants.

Potentiation of Ligand Binding through Cooperative Effects in Monoamine Oxidase B

Crystallographic and biochemical studies have been employed to identify the binding site and mechanism for potentiation of imidazoline binding in human monoamine oxidase B (MAO B). 2-(2-Benzofuranyl)-2-imidazoline (2-BFI) inhibits recombinant human MAO B with a K(i) of 8.3 ± 0.6 μM, whereas tranylcypromine-inhibited MAO B binds 2-BFI with a K(d) of 9 ± 2 nM, representing an increase in binding energy Δ(ΔG) of -3.9 kcal/mol. Crystal structures show the imidazoline ligand bound in a site that is distinct from the substrate-binding cavity. Contributions to account for the increase in binding affinity upon tranylcypromine inhibition include a conformational change in the side chain of Gln(206) and a "closed conformation" of the side chain of Ile(199), forming a hydrophobic "sandwich" with the side chain of Ile(316) on each face of the benzofuran ring of 2-BFI. Data with the I199A mutant of human MAO B and failure to observe a similar binding potentiation with rat MAO B, where Ile(316) is replaced with a Val residue, support an allosteric mechanism where the increased binding affinity of 2-BFI results from a cooperative increase in H-bond strength through formation of a more hydrophobic milieu. These insights should prove valuable in the design of high affinity and specific reversible MAO B inhibitors.

High-level expression and purification of rat monoamine oxidase A (MAO A) in Pichia pastoris: Comparison with human MAO A

The high-level heterologous expression in Pichia pastoris, purification and characterization of recombinant membrane-bound rat liver monoamine oxidase A (MAO A) are described. A 1-L culture of cells produces approximately 700 U of rat MAO A activity. The rat MAO A activity is found in outer mitochondrial membrane of the cell. Using a modification of the human MAO A purification procedure, approximately 200mg of recombinant rat MAO A is purified in a 43% yield and exhibits a molecular weight of approximately 60,000 kDa on SDS-PAGE. The purified enzyme contains a covalently bound FAD and forms a N(5) flavocyanine adduct on inhibition by clorgyline. Edman sequencing shows that the amino terminus of rat MAO A is blocked at an N-terminal threonyl residue. The purified rat enzyme exhibits a higher thermal stability than does purified human MAO A. Compared with human MAO A, rat MAO A oxidizes serotonin or kynuramine with twofold higher k(cat)/K(m) values, oxidizes phenethylamine with a 6.7-fold higher catalytic efficiency and benzylamine with a approximately 40-fold higher catalytic efficiency. Although approximately 90% identical in sequence to human MAO A, rat MAO A is a more efficient catalyst for amine neurotransmitter oxidation.

Impact of Species-Dependent Differences on Screening, Design, and Development of MAO B Inhibitors

The impact of species-dependent differences between human and rat MAO B on inhibitor screening was evidenced for two classes of compounds, coumarin and 5H-indeno[1,2-c]pyridazin-5-one derivatives. All examined compounds have shown a greater inhibitor potency toward human MAO B than toward rat MAO B. Moreover, no correlation was found between human and rat pIC(50) values. These divergences have important implications for the design and development of drugs involved in the MAO B metabolic pathway, suggesting that results obtained using rat enzyme cannot be extrapolated to human CNS, a priori. Indeed, the selection of a hit compound for lead generation could be different using human rather than rat enzyme. Moreover, the influence of substituents on the in vitro inhibition of human MAO B was markedly different between homogeneous series of coumarin and 5H-indeno[1,2-c]pyridazin-5-one derivatives, suggesting different binding modes, a hypothesis clearly supported by molecular docking simulations of inhibitors into the active site of human MAO B.

Three-dimensional structure of human monoamine oxidase A (MAO A): relation to the structures of rat MAO A and human MAO B.

The three-dimensional structure of recombinant human monoamine oxidase A (hMAO A) as its clorgyline-inhibited adduct is described. Although the chain-fold of hMAO A is similar to that of rat MAO A and human MAO B (hMAO B), hMAO A is unique in that it crystallizes as a monomer and exhibits the solution hydrodynamic behavior of a monomeric form rather than the dimeric form of hMAO B and rat MAO A. hMAO A's active site consists of a single hydrophobic cavity of approximately 550 A3, which is smaller than that determined from the structure of deprenyl-inhibited hMAO B (approximately 700 A3) but larger than that of rat MAO A (approximately 450 A3). An important component of the active site structure of hMAO A is the loop conformation of residues 210-216, which differs from that of hMAO B and rat MAO A. The origin of this structural alteration is suggested to result from long-range interactions in the monomeric form of the enzyme. In addition to serving as a basis for the development of hMAO A specific inhibitors, these data support the proposal that hMAO A involves a change from the dimeric to the monomeric form through a Glu-151 --> Lys mutation that is specific of hMAO A [Andrès, A. M., Soldevila, M., Navarro, A., Kidd, K. K., Oliva, B. & Bertranpetit, J. (2004) Hum. Genet. 115, 377-386]. These considerations put into question the use of MAO A from nonhuman sources in drug development for use in humans.

Demonstration of Isoleucine 199 as a Structural Determinant for the Selective Inhibition of Human Monoamine Oxidase B by Specific Reversible Inhibitors

Several reversible inhibitors selective for human monoamine oxidase B (MAO B) that do not inhibit MAO A have been described in the literature. The following compounds: 8-(3-chlorostyryl)caffeine, 1,4-diphenyl-2-butene, and trans,trans-farnesol are shown to inhibit competitively human, horse, rat, and mouse MAO B with K(i) values in the low micromolar range but are without effect on either bovine or sheep MAO B or human MAO A. In contrast, the reversible competitive inhibitor isatin binds to all known MAO B and MAO A with similar affinities. Sequence alignments and the crystal structures of human MAO B in complex with 1,4-diphenyl-2-butene or with trans,trans-farnesol provide molecular insights into these specificities. These inhibitors span the substrate and entrance cavities with the side chain of Ile-199 rotated out of its normal conformation suggesting that Ile-199 is gating the substrate cavity. Ile-199 is conserved in all known MAO B sequences except bovine MAO B, which has Phe in this position (the sequence of sheep MAO B is unknown). Phe is conserved in the analogous position in MAO A sequences. The human MAO B I199F mutant protein of MAO B binds to isatin (K(i) = 3 microM) but not to the three inhibitors listed above. The crystal structure of this mutant demonstrates that the side chain of Phe-199 interferes with the binding of those compounds. This suggests that the Ile-199 "gate" is a determinant for the specificity of these MAO B inhibitors and provides a molecular basis for the development of MAO B-specific reversible inhibitors without interference with MAO A function in neurotransmitter metabolism.

Dramatic Species Differences in the Susceptibility of Monoamine Oxidase B to a Group of Powerful Inhibitors

Oxadiazolones and oxadiazolethiones are potent, reversible competitive inhibitors of MAO B in rat brain mitochondria. We have compared the Ki values of six of these inhibitors toward MAO B from rat, human and beef liver mitochondria, using benzylamine as substrate. Unexpectedly, their inhibitory potency varies by 3 to 4 orders of magnitude between rat and beef liver MAO B, whereas the inhibition of the rat and human liver enzymes is quite similar. Examples are 5-(4-benzyl-oxyphenyl)-3-(2-cyano-ethyl)-1,3,4-oxadiazole-2(3H)- thione, with Ki at 30° of 0.5 nM for rat, 0.8 nM for human, and 1,830 nM for beef liver mitochondria and 5-(4-benzyloxyphenyl)-3-(2-hydroxyethyl)-1,3,4-oxadiazol-2(3H)- one with Ki values of 1.2, 1.1 and 1,320 nM for MAO B from these three sources. Since solubilized and membrane-bound enzymes had similar sensitivities to the inhibitors, the differences seen must arise from differences in the amino acid sequences of the three enzymes.

Intracellular distribution of monoamine oxidase A in selected regions of rat and monkey brain and spinal cord

Monoamine oxidase A and B (MAO A and B; EC 1.4.3.4) are integral proteins of the outer mitochondrial membrane that degrade monoamines including the neurotransmitters norepinephrine, dopamine, and serotonin. In this study, monoclonal antibodies that recognize rat or monkey MAO A were used in immunocytochemical studies to visualize the subcellular localization of this enzyme within neurons in the central nervous system of these species. The regions examined included the locus coeruleus, substantia nigra, spinal cord, and pallidostriatum, which are known to contain MAO A-positive structures. Ultrastructural studies revealed that most MAO A staining was associated with the outer membrane of mitochondria, within the cell bodies, dendrites, axons and terminals. However, some immunoreactive staining for MAO A was also observed in the rough endoplasmic reticulum in the cell bodies. Staining for mitochondrial MAO A in dendrites was observed in terminal fields of the monoamine system, including the spinal cord and the pallidostriatum. The intensity of staining also increased in the subsynaptic density. MAO A was also found associated with mitochondria in ependymal cells lining the fourth ventricle adjacent to the locus coeruleus and in the endothelial cells lining the blood vessels. Localization of MAO A in noradrenergic neurons, ependymal cells, and subsynaptic regions of dendrites in monoamine terminal fields supports the concept that this neurotransmitter-degrading enzyme may play a protective role in the central nervous system.

CDNA cloning and sequencing of rat monoamine oxidase A: Comparison with the human and bovine enzymes

1. 1. The nucleotide and deduced amino acid sequences of rat liver MAO A were determined, and sequence identities among MAO A and B from rat, human and bovine were compared. 2. 2. MAO A from rat exhibited greater than 85% sequence identity with bovine and human MAO A, and 70% identity with rat MAO B. 3. 3. Rat adrenal cDNAs were restriction mapped, partially sequenced and found to be identical to rat liver MAO A, suggesting that these two tissues express the same polypeptide.

Enzymic characteristics of benzylamine oxidase and monoamine oxidase in rat lung

Many studies on various tissues indicate the presence of benzylamine oxidase (BZO), and there are two forms of BZO in rat heart and aorta. Here, we compared enzymic properties of rat lung BZO and MAO and studied the possible existence of two forms of BZO. Subcellular distribution studies revealed that MAO activity was mainly localized in mitochondria, but for BZO, high recoveries of activity were observed in both mitochondria and microsomes. Mitochondrial MAO activity towards tyramine was more highly sensitive to deprenyl than to clorgyline, with a single-sigmoidal curve, indicating MAO-B predominance in the lung. In kinetic analyses, Km values of BZO and MAO for benzylamine in the two fractions were markedly different (about 3-6μM for BZO and 80uM for MAO, respectively). Vmax values of MAO in mitochondria were remarkably higher compared to those in microsomes, but in contrast, BZO values in microsomes were significantly higher. Substrate specificities of these two amine oxidases were extremely different, but those of each enzyme in the two fractions were similar. MAO activities towards various substrates under 20 and 5% of O2 concentrations similarly decreased in proportion to O2 concentration, but those for BZO increased to a similar extent, except towards benzylamine. These results indicate cross-contamination of the two fractions and that BZO might play a role in deaminations of exogenous amines when MAO activity is lowered.

Deamination of 5-methoxytryptamine, serotonin and phenylethylamine by rat MAO in vitro and in vivo

Pretreatment of rats with clorgyline, a selective inhibitor of MAO-A, significantly inhibited the in vivo deamination of intraventricularly administered serotonin (5-HT) and 5-methoxytryptamine (5-MT), but not phenylethylamine (PEA). Pretreatment with d, l-deprenyl, a selective inhibitor of MAO-B, significantly inhibited the in vivo deamination of all three substrates. Brain and liver homogenates from rats pretreated with clorgyline showed a decreased ability to deaminate (in vitro) 5-MT and 5-HT, but not PEA. Homogenates from animals pretreated with d,l-deprenyl showed a decreased capacity to deaminate PEA, but not 5-MT or 5-HT. Clorgyline, when added to brain and liver homogenates, selectively blocked the deamination of 5-MT and 5-HT, but not PEA, whereas, d,l-deprenyl blocked the deamination of PEA without affecting that of 5-MT or 5-HT. In addition, 5-MT was found to be 100 X more potent than PEA at inhibiting the in vitro deamination of 5-HT. These findings suggest that 5-MT and 5-HT are favored substrates for MAO-A in vitro and in vivo. However, in vivo, significant amounts of 5-MT and 5-HT can also be deaminated by MAO-B.