Elucidating the role of radical chemistry in the mechanism of action of monoamine oxidase.

Lead Research Organisation: University of Manchester
Department Name: Life Sciences


The mammalian monoamine oxidases are enzymes found in the outer mitochondrial membrane of all cell types. They catalyse the degradation of neurotransmitters and exogenous alkylamines by the oxidation of these compounds. The human enzymes are major pharmaceutical targets for antidepressants. Inhibitors of the MAO B isoform are used synergistically with L-dopamine in the treatment of Parkinson's disease. Mechanism-based inhibitors are available for clinical use as antidepressants and as neuroprotective drugs. Elevated levels of MAO B induce apoptosis (programmed cell death) in kidney and neuronal cells, and are also associated with plaque astrocytes in the brains of Alzheimer's patients. The anti apoptotic action of a MAO B inhibitor is important in novel Alzheimer treatments. MAO is also implicated in the onset of Parkinson's syndrome through bioactivation of the compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an impurity in many sources of synthetic heroin. The desire to develop more effective and specific enzyme inhibitors has driven programmes aimed at a more detailed understanding of the mechanism of MAO enzymes. Despite these efforts, and the vast and extensive literature on substrate and inhibitor specificities, the mechanism of MAO remains uncertain, although new insights have emerged recently following the availability of the highly purified recombinant forms of human MAO enzymes. MAO is a flavoprotein, and the mechanisms of amine oxidation by flavoproteins in general are poorly understood. A number of mechanisms have been proposed over the years and the debate has centred on the presence /absence of radical species. A key development in recent years has been the elucidation of crystallographic structures for monoamine oxidases A and B, which has re-invigorated the long-standing debate concerning the mechanisms of these enzymes. Our recent identification of a protein-based radical in the MAO enzymes now favours substantially mechanisms based on radical chemistry. In this application we seek to establish the mechanism of these enzymes using advanced spectroscopic and kinetic methods.

Technical Summary

Much of the debate concerning the mechanism of monoamine oxidase has centred on the possible existence of radical species in MAO catalysed oxidation of amines, direct evidence for which, until very recently, was not forthcoming. The prolonged period in which compelling evidence for a radical species was not available has led to several mechanistic proposals. An early proposal based on flavin model reactions, in which 10-phenylisoalloxazine was reacted with methyl phenylglycine, invoked a polar nucleophilic mechanism involving attack of the deprotonated amine substrate at the flavin C4a to form a substrate/flavin C4a adduct. Support for the polar nucleophilic mechanism also came from chemical model studies in reactions of amines with lumiflavins. Of particular interest was the observation of (i) a stable lumiflavin-substrate C4a adduct following reduction of N5-ethyl-3-methyllumiflavinium perchlorate with benzylamine, and (ii) the correlation of the rates of reaction with amine nucleophilicity. The proposed mechanism involves reversible protonation at the N5 atom of 3 methyllumiflavin (3MLF) and nucleophilic addition to form a lumiflavin 4a adduct. The final elimination reaction is proposed to be both acid and base catalysed based on studies of the reactivity of a 4a benzylamino-dihydroflavin adduct. Quantitative structure/activity relationships (QSAR) with MAO B were originally used to support an alternative mechanism in which substrate a-C/H bond cleavage is by direct hydrogen atom transfer to a protein-based non-flavin radical, followed by electron transfer to the flavin. This mechanism requires a relatively stable protein-based radical in the active site of the enzyme. An organic radical species was originally reported in EPR spectra of resting (i.e. flavin in the oxidised state) bovine liver MAO B, but this was attributed later to an artefact of purification of MAO B from bovine liver. More recent studies confirmed the lack of a protein-based radical in highly purified forms of recombinant MAO A and MAO B in the resting state. The lack of an observed radical species in the resting states of MAO A and MAO B therefore seriously questions the validity of this proposed mechanism for the MAO enzymes. In recent work we have demonstrated the presence of a tyrosyl radical in partially reduced monoamine oxidase, which provides the key missing link in support of radical chemistry in these enzymes. The work described in the current application seeks to demonstrate the kinetic competence of this radical species using advanced spectroscopic and kinetic methods. The aim is to provide strong support for the aminyl radical cation mechanism for amine oxidation by MAO enzymes. The work will substantially advance our understanding of mechanism in this important enzyme family and remove much of the controversy and confusion regarding the mechanism of catalysis that is currently present in the literature.
Description We were able to provide unequivocal evidence for the importance of tyrosyl radicals in monoamine oxidases, which lends further support to the importance of radical species in flavoprotein amine oxidations in general. We also highlighted for the first time the issue of radical 'escape' from the active site which has important implications in cellular protection against radical species, especially when targeting this enzyme therapeutically (MAO is a major drug target). We were also able to correct important mechanistic information, previously incorrect in the literature, on the nature of the protonic states for substrate and catalytic residues in the active site.
Sectors Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology,Other

Description The more comprehensive mechanistic understanding will now underpin mechanism based inhibitor design of amine oxidases in general, which are major targets in the pharmaceutical industry.
First Year Of Impact 2008
Sector Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology,Other
Impact Types Cultural

Description EMFBRT
Amount £208,792 (GBP)
Funding ID BFT 10/35 
Organisation The EMF Biological Research Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2010 
End 10/2014
Title Freeze-Quench 
Description Rapid mixing technique to freeze quench intermediates for EPR studies. 
Type Of Material Improvements to research infrastructure 
Provided To Others? No  
Impact Publications