Characterization of Euphorbia characias Latex Amine Oxidase1

Abstract

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 ؋ 10 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 quinonecontaining amine oxidases. This hexapeptide contains the tyrosinyl residue that can be modified into the cofactor 6-hydroxydopa quinone.Copper-amine oxidases (amine oxygen oxidoreductase deaminating, copper containing; EC 1.4.3.6) are widespread enzymes oxidizing primary amines with the formation of the corresponding aldehyde, ammonia, and hydrogen peroxide: R-CH 2 -NH 2 ϩO 2 ϩH 2 O3 R-CHOϩNH 3 ϩH 2 O 2 .These enzymes are ubiquitous in nature, occurring in microorganisms (fungi and bacteria; Cooper et al., 1992), plants (Medda et al., 1995a), and mammals (McIntire and Hartmann, 1993). The crystal structures of copper-amine oxidases from Escherichia coli (Parson et al., 1995) and pea seedlings (Kumar et al., 1996) were recently determined. Amine oxidases are homodimers of 70-to 95-kD subunits. Each subunit contains a tightly bound Cu(II) center that is essential for the enzyme redox activity (Dooley et al., 1991;Medda et al., 1995b), and TPQ is formed by a posttranslational modification from a tyrosinyl residue in a copperdependent, autocatalytic reaction (Janes et al., 1990;

Electroactive Centers inEuphorbiaLatex and Lentil Seedling Amine Oxidases

“…3,5) Only high-quality protein was used, on the basis of 2 AE 0:1 titratable TPQ per dimer. The concentration of the enzymes was determined using " 278 ¼ 2:…”

“…for LSAO 3) and ELAO 5) respectively. Copper-depleted enzymes were prepared as previously described.…”

“…The best known and studied are those from seedlings of the pulses lentil (Lens esculenta; LSAO) 3) and pea (Pisum sativum; PSAO), 4) and from latex of Euphorbia characias, a perennial Mediterranean shrub (ELAO). 5) Plant Cu/TPQ amine oxidases catalyze the oxidation of the primary amino group of mono-, di-and polyamines withe formation of the corresponding aldehydes, ammonia, and hydrogen peroxide, subtracting two electrons from amines and transferring them to molecular oxygen. These enzymes can flip-flop from oxidized to reduced states through chemical reaction with amine, and come up oxidized by molecular oxygen, 6,7) as in equations 1 and 2:…”

Catabolic pathways for arginine and methylated arginines by plant and mammalian copper amine oxidases

“…These enzymes are characterized by the presence of two redox centers, a divalent copper and the quinone of 2,4,5-trihydroxyphenylalanine (TPQ) derived by a post-synthetic modification of a tyrosinyl residue of the protein [7,8]. Plant CAOs from various species have been purified to homogeneity and characterized, the best known and studied being those from lentil (Lens esculenta) [9] and pea (Pisum sativum) [10] and from latex of the shrub Euphorbia characias [11]. In mammals, the best known enzymes are those from pig kidney and from bovine serum [12][13][14].…”

Copper/topaquinone-containing amine oxidase from lentil seedlings and bovine plasma: Catalytic mechanism and energetic domains

“…Plant amine oxidases from various species of several families have been purified to homogeneity and characterized. Among these enzymes, the best known and studied are those isolated from seedlings of the pulses lentil (Lens esculenta) [4], pea (Pisum sativum) [5] and Euphorbia characias (a shrub) latex [6]. b.…”