The oxidation of N-substituted aromatic amines by horseradish peroxidase.
The mechanism of N-dealkylation by peroxidases of the Ca2+ indicator quin2 and analogs was investigated and compared with the mechanism of N-dealkylation of some N-methyl-substituted aromatic amines. Nitrogen-centered cation radicals were detected by ESR spectroscopy for all the compounds studied. Further oxidation of the nitrogen-centered cation radicals, however, was dependent upon the structure of the radical formed. In the case of quin2 and analogs, a carbon-centered radical could be detected using the spin trap 5,5-dimethyl-1-pyrroline N-oxide. By using the spin trap 2-methyl-2-nitrosopropane (tert-nitrosobutane), it was determined that the carbon-centered radical was formed due to loss of a carboxylic acid group. This indicated that bond breakage most likely occurred through a rearrangement reaction. Furthermore, extensive oxygen consumption was detected, which was in agreement with the formation of carbon-centered radicals, as they avidly react with molecular oxygen. Thus, reaction of the carbon-centered radical with oxygen most likely led to the formation of a peroxyl radical. The peroxyl radical decomposed into superoxide that was spin trapped by 5,5-dimethyl-1-pyrroline N-oxide and an unstable iminium cation. The iminium cation would subsequently hydrolyze to the monomethyl amine and formaldehyde. In the case of N-methyl-substituted aromatic amines, carbon-centered radicals were not detected during the peroxidase-catalyzed oxidation of these compounds. Thus, rearrangement of the nitrogen-centered radical did not occur. Furthermore, little or no oxygen consumption was detected, whereas formaldehyde was formed in all cases. These results indicated that the N-methyl-substituted amines were oxidized by a mechanism different from the mechanism found for quin2 and analogs.
Van der Zee
J
J
Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Duling
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DR
Mason
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Eling
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D000814:Aniline Compounds; D004533:Egtazic Acid / Q000031:analogs & derivatives; D004578:Electron Spin Resonance Spectroscopy; D005557:Formaldehyde / Q000032:analysis; D005609:Free Radicals; D006735:Horseradish Peroxidase / Q000378:metabolism*; D007700:Kinetics; D009603:Nitroso Compounds; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D010544:Peroxidases / Q000378:metabolism*; D013113:Spin Labels; D013379:Substrate Specificity; D014052:Toluidines
D000814:Aniline Compounds; D005609:Free Radicals; D009603:Nitroso Compounds; D013113:Spin Labels; D014052:Toluidines; D005557:Formaldehyde; D004533:Egtazic Acid; C015157:N,N-dimethylaniline; D006735:Horseradish Peroxidase; D010544:Peroxidases; C034526:tert-nitrosobutane; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; C015835:dimethyl-4-toluidine