Intermediates in the catalytic cycle of copper-quinoprotein amine oxidase from Escherichia coli.
Investigations on the reduction of copper quinoprotein amine oxidases (EC 1.4.3.6) by substrate indicate that the nature of the reduced enzyme species formed varies, as judged from the spectroscopic data reported in the literature for different enzymes and substrates. The availability of substantial amounts of overproduced, homogeneous Escherichia coli amine oxidase (ECAO) enabled us to investigate this aspect with a number of different approaches: quantitative titration of enzyme with substrate, stopped-flow kinetic spectrophotometry (anaerobic and semianaerobic), EPR spectroscopy of stable intermediates in the catalytic cycle, and conversions with H2O2 as the oxidant. Reduction of ECAO by a variety of substrates led to spectra (UV/Vis, EPR) identical to those that have been ascribed to the semiquinone form of the topaquinone cofactor. The extent of semiquinone formation was enhanced in the presence of KCN, but the properties of the artificially induced semiquinone were different from those of the spontaneously induced one, as shown by the spectroscopic data and the reactivity toward O2 and H2O2. On titrating ECAO at high concentrations with substrate, evidence was obtained that disproportionation takes place of the semiquinone formed, the reaction most probably proceeding via intermolecular electron transfer, leading to a topaquinone- and Cu1+-containing enzyme species that is able to perform substrate conversion. The latter, as well as OH*, is probably also formed when H2O2 replaces O2 as oxidant, explaining why substrate conversion with concomitant enzyme inactivation occurs under this condition. Formation of the semiquinone was always preceded by that of a hitherto unknown species with an absorbance maximum at 400 nm. The structure proposed for this species is a protonated form of the aminoquinol cofactor, the Zwitter ionic structure being stabilized by amino acid residues in the active site having opposite charges. Based on the properties observed and the moment of appearance during conversions, a proposal is made for the sequence in which the three reduced enzyme species convert into each other.
Steinebach
V
V
Kluyver Laboratory of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
de Vries
S
S
Duine
J A
JA
D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry* / Q000378:metabolism*; D000693:Anaerobiosis; D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007700:Kinetics; D010627:Phenethylamines / Q000494:pharmacology; D011190:Potassium Cyanide / Q000494:pharmacology; D011487:Protein Conformation; D013053:Spectrophotometry; D013997:Time Factors
D010627:Phenethylamines; D006861:Hydrogen Peroxide; D006631:Amine Oxidase (Copper-Containing); D011190:Potassium Cyanide