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  1. Handbook of Flavoproteins, Volume 1: Oxidases, Dehydrogenases and Related Systems
  2. Upcoming Events
  3. Multi-volumed work
  4. Russ Hille: Books

Kavitha , Sri Lakshmi Narasimha Published The biologically inspired Neural Networks are computer programs designed to simulate the way in which the human brain processes information. Neural network gather their knowledge by detecting the patterns and relationships in data and learn through experience. Neural network was not only used for classification of physiologically active substances but also for solving the quantitative structure activity relationship problem. View PDF. Save to Library.

Create Alert. Share This Paper. Figures and Tables from this paper. Figures and Tables. Their three-dimensional structures have been determined over the past de- cade and have led to a more definitive understanding of their functions at a molecular level. Highly specific reversible and irre- versible inhibitors have been identified for MAO B and our understanding of this en- zyme exceeds current detailed insights of MAO A.

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An extensive literature exists on both enzymes and this review summarizes the molecular properties of both purified and membrane-bound forms of the enzymes. Recent developments have shown the enzymes are topologically situated on distinct faces of the mitochondrial outer mem- brane and that they exhibit quite different behaviors when p-substituted benzylamine analogues are used as mechanistic probes. The impact of these differential structural and functional properties on their biological roles remain open questions in the field.

In addition, these enzymes degrade ingested amines e. This finding sparked considerable investigations into various MAO inhibitors by both academic and pharmaceutical laboratories to develop drugs that could be used as anti-depressants and active research in this field continues [1]. Unequivocal demonstration that the two forms are separate gene products came from delineations of their respective gene sequences [6] and location of those genes on the human X-chromosome [7]. Further work has identified both enzymes are bound to the mitochon- drial outer membrane in all tissues although the tissue distribution of MAO A and MAO B differ among mammals investigated [11].

Therefore, both enzymes are the focus of contin- ued investigations for their roles in these age-related diseases. A breakthrough in this endeavor came with the development of expression systems for the production and purification of both human MAO B and MAO A in reagent quantities. Expression and purification of the rat and zebrafish MAOs allow for comparative functional studies with the human enzymes to facilitate the interpretation of results from animal models for drug develop- ment.

Human MAO B was the first of the two enzymes to be crystallized and the structure determined [23]. This difference in gating residues is proposed to account for differences in inhibitor specificities observed on comparison of bovine and human MAO B [24,25]. A similar strain and aromatic cage is also present in MAO A.

The last 20 residues of this helix are too disordered to be visible in the structure. The protein structure is represented in light blue, the protein chains representing the membrane binding domain in green and the dipartite active site cavity as pink transparent surface.

The FAD moiety is depicted in yellow, with nitrogen, oxygen, sulfur and phosphor atoms in blue, red, green and magenta, respectively. B Close- up representation of the dipartite active site cavity in human Monoamine Oxidase B. This result raised the interesting ques- tion as to whether there is any functional importance of this difference in oligomeric forms between human MAO A and MAO B. Distances determined for MAO A and for MAO B were found to be consistent with dimeric structures for both enzymes in the mito- chondrial outer membrane.

Handbook of Flavoproteins, Volume 1: Oxidases, Dehydrogenases and Related Systems

The human MAO A monomer appears to crystallize more readily whereas it is the dimeric form of the rat enzyme that favors crystallization. These data also demonstrate that the structures of the membrane-bound forms of MAO A and MAO B exhibit distances identical with those found in their respective crystalline forms. One added benefit from studies of the TEMPO-substituted pargylines as active site MAO probes is that they are unable to cross the outer membrane in intact mitochondria [31].

Us- ing these inhibitors as probes in conjunction with protease inactivation experiments, the membrane topologies of human and rat MAO A and B were investigated. Phe Fig. The molecular basis for this differential topology and influences on inhibitor interactions is under ongoing investigation. The mechanism for this attachment appears to be autocatalytic by analogy with other flavoenzyme systems investigated. The only flavin analogue tested that was not covalently incorporated is 5-deaza FAD. No restoration of activity was observed on the addition of either FMN or riboflavin.

The apoenzyme is unstable to solubilization from its membrane environment; however it does bind a number of FAD analogues with Kd values in the 50— nM range. These studies demonstrate that covalent binding is not essential for MAO activity but that the presence of a covalent FAD stabilizes the enzyme structure permitting solubilization in detergents and purification. MAO B has a higher specificity for phenethylam- ine or benzylamine so these are the substrates of choice for estimating MAO B activity.

Tipton and Singer have pointed out pitfalls in this assay procedure [40] and most current assay procedures utilize tyramine as substrate and the fluorometric detection of H2O2 with the horseradish peroxidase-coupled Amplex Red assay [41]. This assay is convenient and sensitive but does have limitations.

It cannot be used with catechol-type substrates such as serotonin or dop- amine since these compounds also are substrates for horseradish peroxidase. Thus, caution is needed when using this assay procedure to measure MAO inhibition since any inhibitor with catechol or aniline functionalities may also react with the peroxidase. Another caution to be observed when using the fluorescence assay is that oxidation of the resorufin product to a non-fluorescent resazurin by horseradish peroxidase or to other non-fluorescent spe- cies has also been observed when initial rate determinations of more than 3—4 min duration are used [43].

The fact that no reports of any resorufin inhibition of MAO have been noted in publications may reflect the low concentration of product formed in initial rate assays.

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Temperature effects on MAO B activity can therefore exhibit large changes in rates since the [O2] in aqueous solutions is highly temperature dependent. Extensive kinetic work on bovine MAO B [44,45] has shown the enzyme follows a ternary complex i. The measured second-order rate constants for O2 oxidation of the free, reduced enzyme and for the reduced enzyme-protonated imine complex are shown in red lettering. Most of the available data with MAO A support the enzyme following a ternary complex mechanism as well since the presence of substrate enhances the rate of oxidation of the reduced form of the enzyme [46].

The most reasonable explanation is the electrostatic stabilization of the formation of the anionic superoxide intermediate resulting from the single electron reduction of O2 as found to be the role of a His residue in glucose oxidase [47]. Direct evidence for formation of the protonated imine as the initial product formed at a rate identical with that of flavin reduction and subsequent release into the medium is found with studies on the MAO B oxidation of para-dimethylamino-benzylamine [48].

This analogue exhibits spectral properties that allow detection of the protonated form of the imine as reaction product and permits measurement of its rate of hydrolysis, which occurs non-enzymatically in the solvent rather than in the catalytic site. Similar results are also found with MAO A showing that imine hydrolysis occurs after its release from the enzyme. Similar pathways have been shown for other flavoprotein oxidases as well. It is well known that D-amino acid oxidase follows either a binary or ternary complex mechanism depending on the nature of the amino acid substrate.

Other more complex mechanisms such as that suggested by Ramsay et al. In their published scheme, it is suggested that a possible cause for finding deviations from Michaelis-Menten behavior in MAO B and apparently not in MAO A, in contrast to their prior work is due to displacement of imine product from the reduced enzyme by substrate resulting in the differential expression of steady state parameters Vmax and KM values for both the oxidized and reduced enzyme species.

These deviations from Michaelis-Menten behavior were observed using the Amplex-Red coupled assay with fluorescent resorufin product detection using well plate reader assays which allow for a large number of assays to improve the errors commonly observed in the measurement of rates in single catalytic assays. What is not considered in these studies is the inclusion of error from for- mation of non-fluorescent resorufin products [42] or the possibility of any substrate inhibi- tion.

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Extensive data supporting reoxidation of the reduced enzyme-imine complex by O2 [44,45] in benzylamine oxidation assays would argue that very little if any free reduced MAO B is available in the steady state to bind to substrate. A better case could be made for such catalytic complexes involving the free reduced form of MAO B interacting with the substrate using phenethylamine since free, reduced enzyme occurs as an intermedi- ate during catalysis [44].

Thus, MAO B is susceptible to substrate inhibition, which becomes more observable at high pH values and provides a further complication in the interpretation of MAO B steady state kinetic data. Such behavior is not observed with MAO A and probably reflects the monopartite nature of its active site cavity. The mechanism of amine oxidation and flavin reduction in MAO has been a con- troversial topic with three possibilities suggested in the literature. Silverman and co- workers have suggested the initial step to be a 1-electron oxidation of the amine by the flavin to form an aminium cation radical and a flavin radical i.

The major experimental support for this mechanism is the observation of cyclopropyl ring open- ing of the mechanism-based inhibitor tranylcypromine concomitant with formation of a flavin C 4a adduct. Supporting evidence for a single electron transfer mechanism is weak and a number of experiments argue against it. No effect of magnetic field on the reduction rate of bovine MAO B is observed [59] as might be expected for the reaction of a radical pair. Rapid scan stopped flow experiments with substrate analogues where C-H bond cleavage is rate limiting large Dk values provide no spectral evidence for any flavin semiquinone intermediates.

With the demonstration that D-amino acid oxidase follows a hydride ion transfer mechanism for flavin reduction [60], Fitzpatrick and co-workers have argued that flavin- dependent amine oxidases including MAO function by hydride ion transfer steps since no spectral intermediates are observed in a number of flavoenzymes catalyzing amine oxidation [61]. The reaction products reduced flavin and protonated imine are shown in red. Bovine MAO B oxidation of benzylamine analogues show no contributions of elec- tronic effects from para-substituents [45] and has been interpreted [62] as being due to the conformation of the bound substrate in the catalytic site preventing orbital overlap for transmission of electronic effects.

Recent 15N kinetic isotope effect measurements of human MAO B oxidation of 1H and 2H benzylamine analogues [65] show C-H bond cleavage is not concerted with the change in bond order to N in the transition state, which argues that a Hydride ion mechanism for C-H bond cleavage, if it does occur, is not a concerted step in MAO B catalysis. The available mechanistic data support a polar nucleophilic mechanism for MAO A catalysis [62].

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Whether this assumption is correct can only be defined by detailed comparative mechanistic experiments. By analogy with the role of a His in glucose oxidase [47] that stabilizes the initial formation of a radical pair of superoxide anion and flavin semiquinone, the presence of imine or amine in the active sites of MAO B or MAO A could perform a similar stabilization resulting in enhanced rates. This structure is also seen in a number of flavoprotein oxidases and may have an important role in the reaction.

Jorns and colleagues found a dramatic decrease in O2 reactivity of monomeric sarcosine oxidase [68] on mutagenesis of this Lys to a Met. Similar experiments with other flavoprotein oxidases have resulted in mixed results [69] and, therefore, no general role of this Lys residue in flavoprotein oxidases can be formu- lated regarding its role in the reaction with O2.

The most salient aspects to consider by the interested reader is that the tissue specific expression of MAO is altered along development, with MAO B levels increasing 4-fold in neuronal tissue while cardiac levels of MAO A increase 9-fold on aging. This is in addition to their better known involvement in disease states such as depression.

The therapeutic potential of monoamine oxidase inhibitors. Nat Revs Neurosci ;— The covalently- bound flavin of hepatic monoamine oxidase. Eur J Biochem ;—7. Arch Biochem Biophys ;— On the role and formation of covalently bound flavin cofactors. FEBS J ;— Molecular characterization of monoamine oxidase in zebrafish Danio rerio.

Comp Biochem Phys B ;— Comp Biochem Physiol Part B. Developmental aspects of the monoamine degrad- ing enzyme monoamine oxidase. Dev Pharmac Ther ;— J Neurosci ;— The effect of age on the activity and molecular properties of human brain monoamine oxidase. J Neural Transm. Age- dependent increase in hydrogen peroxide production by cardiac monoamine oxidase A in rats. High-level expression of human liver mono- amine oxidase B in Pichia pastoris. Prot Exp Purif ;— High level expression of human liver monoamine oxidase A in Pichia pastoris.