Inactivation of copper-containing amine oxidases by turnover products.
For bovine serum amine oxidase, two different mechanisms of substrate-induced inactivation have been proposed. One consists of a slow oxidation by H2O2 of a conserved residue in the reduced enzyme after the fast turnover phase [Pietrangeli, P., Nocera, S., Fattibene, P., Wang, X.T., Mondovì, B. & Morpurgo, L. (2000) Biochem. Biophys. Res. Commun.267, 174-178] and the other of the oxidation by H2O2 of the dihydrobenzoxazole in equilibrium with the product Schiff base, during the catalytic cycle [Lee, Y., Shepard, E., Smith, J., Dooley, D.M. & Sayre, L.M. (2001) Biochemistry40, 822-829]. To discriminate between the two mechanisms, the inactivation was studied using Lathyrus cicera (red vetchling) amine oxidase. This, in contrast to bovine serum amine oxidase, formed the Cu+-semiquinolamine radical with a characteristic UV-vis spectrum when oxygen was exhausted by an excess of any tested amine in a closed cuvette. The inactivation, lasting about 90 min, was simultaneous with the radical decay and with the formation of a broad band (shoulder) at 350 nm. No inactivation occurred when a thousand-fold excess of amine was rapidly oxidized in an L. cicera amine oxidase solution stirred in open air. Thus, the inactivation is a slow reaction of the reduced enzyme with H2O2, following the turnover phase. Catalase protected L. cicera amine oxidase from inactivation. This effect was substrate-dependent, varying from full protection (benzylamine) to no protection (putrescine). In the absence of H2O2, a specific inactivating reaction, without formation of the 350 nm band, was induced by some aldehydes, notably putrescine. Some mechanisms of inactivation are proposed.
Pietrangeli
Paola
P
Department of Biochemical Sciences A. Rossi Fanelli and C.N.R. Institute of Molecular Biology and Pathology, University of Rome La Sapienza, Rome, Italy. paola.pietrangeli@uniroma1.it
Nocera
Stefania
S
Federico
Rodolfo
R
Mondovì
Bruno
B
Morpurgo
Laura
L
D006631:Amine Oxidase (Copper-Containing) / Q000037:antagonists & inhibitors*; D000818:Animals; D002417:Cattle; D003300:Copper / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000494:pharmacology; D007700:Kinetics; D027807:Lathyrus / Q000201:enzymology*; D010936:Plant Extracts / Q000302:isolation & purification / Q000494:pharmacology*; D010940:Plant Proteins / Q000037:antagonists & inhibitors; D011073:Polyamines / Q000494:pharmacology; D013053:Spectrophotometry; D013095:Spermidine / Q000494:pharmacology; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D010936:Plant Extracts; D010940:Plant Proteins; D011073:Polyamines; D003300:Copper; D006631:Amine Oxidase (Copper-Containing); D013095:Spermidine