Kinetic properties of recombinant MAO-A on incorporation into phospholipid nanodisks.
Recent structural studies of human monoamine oxidase A (MAO-A) suggest the entrance to the active site is positioned near the surface of the mitochondrial outer membrane (Colibus et al., 2005). To determine the influence of the phospholipid bilayer on the structure and catalytic properties of MAO in a defined system, we have incorporated the recombinant protein into phospholipid 'nanodiscs' which have been developed by Stephen G. Sligar's group (Denisov et al., 2004). Purified MAO-A incorporates into pre-formed nanodiscs which are approximately 10 nm in diameter and exhibit the thickness expected for a phospholipid bilayer. Nanodisc assemblies of MAO-A are water-soluble, yield increased enzyme stability relative to detergent solutions, are catalytically active, and reactive with acetylenic inhibitors. As compared to detergent-based systems, the catalytic efficiencies (k (cat)/K (m)) of amine oxidation appear to be greater. Also, nanodisc bound MAO-A binds various inhibitors with K (i) values that are 2-4 fold lower than MAO-A in reduced Triton X-100 solutions. Taken together, these data suggest that the membrane environment affects MAO-A catalytic properties for both substrates and reversible inhibitors.
Cruz
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Department of Biochemistry, School of Medicine, Emory University, Atlanta, GA 30322, USA. fcruz2@emory.edu
Edmondson
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D000818:Animals; D020134:Catalytic Domain / Q000502:physiology; D004791:Enzyme Inhibitors / Q000737:chemistry; D004795:Enzyme Stability / Q000502:physiology; D006801:Humans; D007700:Kinetics; D008563:Membrane Lipids / Q000737:chemistry* / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D008995:Monoamine Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D049329:Nanostructures / Q000737:chemistry*; D017830:Octoxynol / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004791:Enzyme Inhibitors; D008563:Membrane Lipids; D010743:Phospholipids; D011994:Recombinant Proteins; D017830:Octoxynol; D008995:Monoamine Oxidase