Catalytic turnover of substrate benzylamines by the quinone-dependent plasma amine oxidase leads to H2O2-dependent inactivation: evidence for generation of a cofactor-derived benzoxazole.
Incubation of bovine plasma amine oxidase (BPAO) with benzylamine and various p-substituted analogues results in a time-dependent inactivation that is attributable to buildup of the H(2)O(2)-turnover product on the basis of protection afforded by coincubation with catalase. The mechanism of inactivation is distinct from that effected by H(2)O(2) itself, which requires higher concentrations. Solution studies using models for the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor reveal a loss of catalytic activity arising from oxidation of the dihydrobenzoxazole tautomer of the product Schiff base, that competes with hydrolytic release of benzaldehyde product. The resulting stable benzoxazole exhibits a characteristic absorption depending on the nature of the benzylamine p-substituent. For benzylamine itself, the model benzoxazole absorbs at 313 nm, in an area of strong absorption by the enzyme, whereas for 4-nitrobenzylamine, the absorption of the model benzoxazole is sufficiently red-shifted (at 365 nm) to be discerned above the background enzyme absorption. Inactivation of BPAO by 4-nitrobenzylamine is accompanied by loss of the resting TPQ anion absorption at 480 nm concomitant with generation of a new absorption near 360 nm. Resonance Raman spectra of the inactivated enzyme show a close correspondence with those for the model 4-nitrobenzylamine-derived benzoxazole. Substrate-dependent inactivation is also observed for the other two mammalian enzymes examined, equine plasma amine oxidase and human kidney amine oxidase. Catalase provides complete protection in these instances as well. Benzoxazole formation may constitute a common mechanism of inactivation of quinone-dependent amine oxidases by normal substrates in vitro if the product H(2)O(2) is permitted to accumulate. More importantly, the results suggest that the benzoxazole inactivation pathway may be important physiologically and may have influenced the distribution of amine oxidases and catalase in cells.
Lee
Y
Y
Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Shepard
E
E
Smith
J
J
Dooley
D M
DM
Sayre
L M
LM
D006631:Amine Oxidase (Copper-Containing) / Q000037:antagonists & inhibitors / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D016227:Benzoquinones / Q000737:chemistry* / Q000378:metabolism; D001583:Benzoxazoles / Q000737:chemistry* / Q000378:metabolism; D001596:Benzylamines / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D002384:Catalysis; D002417:Cattle; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000737:chemistry* / Q000378:metabolism; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D013379:Substrate Specificity
D016227:Benzoquinones; D001583:Benzoxazoles; D001596:Benzylamines; D004791:Enzyme Inhibitors; C004532:quinone; D006861:Hydrogen Peroxide; D006631:Amine Oxidase (Copper-Containing)