Characterization of Euphorbia characias Latex Amine Oxidase¹

Alessandra Padiglia^*, Rosaria Medda, Anita Lorrai, Barbara Murgia, Jens Z. Pedersen, Alessandro Finazzi Agró, and Giovanni Floris

Department of Biochemistry and Human Physiology, University of Cagliari, Cagliari, Italy (A.P., R.M., A.L., B.M., G.F.); Department of Chemistry, Odense University, Odense, Denmark (J.Z.P.); and Department of Experimental Medicine, University of Rome "Tor Vergata," Rome, Italy (A.F.A.)

A copper-containing amine oxidase from the latex of Euphorbia characias was purified to homogeneity and the copper-free enzyme obtained by a ligand-exchange procedure. The interactions of highly purified apo- and holoenzyme with several substrates, carbonyl reagents, and copper ligands were investigated by optical spectroscopy under both aerobic and anaerobic conditions. The extinction coefficients at 278 and 490 nm were determined as 3.78 × 10⁵ M¹ cm¹ and 6000 M¹ cm¹, respectively. Active-site titration of highly purified enzyme with substrates and carbonyl reagents showed the presence of one cofactor at each enzyme subunit. In anaerobiosis the native enzyme oxidized one equivalent substrate and released one equivalent aldehyde per enzyme subunit. The apoenzyme gave exactly the same 1:1:1 stoichiometry in anaerobiosis and in aerobiosis. These findings demonstrate unequivocally that copper-free amine oxidase can oxidize substrates with a single half-catalytic cycle. The DNA-derived protein sequence shows a characteristic hexapeptide present in most 6-hydroxydopa quinone-containing amine oxidases. This hexapeptide contains the tyrosinyl residue that can be modified into the cofactor 6-hydroxydopa quinone.

Plant Physiol. (1998) 117: 1363-1371
Copyright Clearance Center:   0032-0889/98/117//09
© 1998 American Society of Plant Physiologists

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