CHARACTERIZATION OF EUPHORBIA-CHARACIAS LATEX AMINE OXIDASE

A copper-containing amine oxidase from the latex of Euphorbia characia s was purified to homogeneity and the copper-free enzyme obtained by a ligand-exchange procedure. The interactions of highly purified apo- a nd holoenzyme with several substrates, carbonyl reagents, and copper l igands were investigated by optical spectroscopy under both aerobic an d anaerobic conditions. TI le extinction coefficients at 278 and 490 n m were determined as 3.78 x 10(5) M-T cm(-1) and 6000 M-1 cm(-1), resp ectively. Active-site titration of highly purified enzyme with substra tes and carbonyl reagents showed the presence of one cofactor at each enzyme subunit. In anaerobiosis the native enzyme oxidized one equival ent substrate and released one equivalent aldehyde per enzyme subunit. The apoenzyme gave exactly the same 1:1:1 stoichiometry in anaerobios is and in aerobiosis. These findings demonstrate unequivocally that co pper-free amine oxidase can oxidize substrates with a single half-cata lytic cycle. The DNA-derived protein sequence shows a characteristic h exapeptide present in most 6-hydroxydopa quinone-containing amine oxid ases. This hexapeptide contains the tyrosinyl residue that can be modi fied into the cofactor 6-hydroxycaopa quinone.