Characterization of Euphorbia characias latex amine oxidase.
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 x 10(5) M-1 cm-1 and 6000 M-1 cm-1, 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.
Department of Biochemistry and Human Physiology, University of Cagliari, Cagliari, Italy.
A
A
Padiglia
R
R
Medda
A
A
Lorrai
B
B
Murgia
J Z
JZ
Pedersen
A
A
Finazzi Agró
G
G
Floris
D006631:Amine Oxidase (Copper-Containing); D000595:Amino Acid Sequence; D001483:Base Sequence; D018744:DNA, Plant; D019665:Euphorbiaceae / Q000201:enzymology*; D005609:Free Radicals; D008969:Molecular Sequence Data; D008970:Molecular Weight; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics / Q000378:metabolism*; D012663:Semicarbazides / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013774:Tetranitromethane / Q000378:metabolism
D018744:DNA, Plant; D005609:Free Radicals; D012663:Semicarbazides; C010059:carbamylhydrazine; D006631:Amine Oxidase (Copper-Containing); D000587:Oxidoreductases Acting on CH-NH Group Donors; D013774:Tetranitromethane