The reaction mechanism of copper amine oxidase: detection of intermediates by the use of substrates and inhibitors.
Intermediate states in the catalytic mechanism of lentil copper amine oxidase have been investigated by ESR and optical spectroscopy. Using highly purified apo- and holoenzyme in combination with a poor substrate and a range of inhibitors, under both aerobic and anaerobic conditions, the single steps of the reaction mechanism can be slowed down or 'frozen' completely. In this way, a sequence of six intermediate species in the catalytic cycle has been established. Oxidative deamination of p-(dimethylamino)benzylamine is 5 x 10(5) times slower than for putrescine; the rate-limiting step is shown to be release of the aldehyde product. This process is not affected in the apoenzyme, but subsequent intramolecular electron transfer to form the characteristic free radical intermediate is completely blocked, and the apoenzyme is trapped as an aminoresorcinol species. Classic hydrazine and hydrazide inhibitors bind to the 6-hydroxydopa cofactor in the same way as active substrates, but rearrangements lead to formation of stable intermediate adducts at the step preceding release of aldehyde. The semicarbazide-6-hydroxydopa adduct is shown to bind simultaneously to Cu(II), providing the first direct evidence for localization of 6-hydroxydopa close to the copper site.
Medda
R
R
Institute of Biological Chemistry, University of Cagliari, Italy.
Padiglia
A
A
Pedersen
J Z
JZ
Rotilio
G
G
Finazzi Agrò
A
A
Floris
G
G
D000332:Aerobiosis; D006631:Amine Oxidase (Copper-Containing) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000693:Anaerobiosis; D000814:Aniline Compounds / Q000378:metabolism; D001051:Apoenzymes / Q000378:metabolism*; D001596:Benzylamines / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism*; D003300:Copper / Q000378:metabolism; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D007887:Fabaceae / Q000201:enzymology; D008667:Metalloproteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010946:Plants, Medicinal; D011700:Putrescine / Q000378:metabolism; D012663:Semicarbazides / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity
D000814:Aniline Compounds; D001051:Apoenzymes; D001596:Benzylamines; D003067:Coenzymes; D008667:Metalloproteins; D012663:Semicarbazides; C035815:4-dimethylaminobenzylamine; C001388:6-hydroxydopa; D004295:Dihydroxyphenylalanine; D003300:Copper; D006631:Amine Oxidase (Copper-Containing); D011700:Putrescine