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;
The reaction with substrates and carbonyl reagents of native lentil Cu-amine oxidase and its modified forms, i.e. Cu-fully-depleted, Cu-half-reconstituted, Cu-fully-reconstituted, Co-substituted, Ni-substituted and Zn-substituted, has been studied. Upon removal of only one of the two Cu ions, the enzyme loses 50% of its enzymatic activity. Using several substrates, Co-substituted lentil amine oxidase is shown to be active but the k(c) value is different from that of native or Cu-fully-reconstituted enzyme, while K(m) is similar. On the other hand, the Ni- and Zn-substituted forms are catalytically inactive. Enzymatic activity measurements and optical spectroscopy show that only in the Co-substituted enzyme is the organic cofactor 6-hydroxydopa quinone reactive and the enzyme catalytically competent, although less efficient. The Co-substituted amine oxidase does not form the semiquinone radical as an intermediate of the catalytic reaction. While devoid or reduced of catalytic activity, all the enzyme preparations are still able to oxidise two moles of substrate and to release two moles of aldehyde per mole of dimeric enzyme. The results obtained show that although Co-substituted amine oxidase is catalytically competent, copper is essential for the catalytic mechanism.
Copper amine oxidase was found to be inhibited in a complex way by small alkali metal ions. Classic enzyme kinetic studies showed that Li 1 and Na 1 were weak noncompetitive inhibitors, whereas the larger alkali metals K 1 , Rb 1 and Cs 1 were not inhibitors. However, freezing in the presence of Na 1 or Li 1 surprisingly resulted in complete and irreversible inactivation. In the case of Li 1 , it was possible to show that one ion per subunit was retained permanently in the inactivated enzyme, suggesting a structural rearrangement. The mechanism of inhibition was studied using a wide range of spectroscopic and analytic techniques. Only minor changes in the protein structure could be detected, except for a significant change in the geometry of the copper site. The unique topaquinone cofactor was apparently functional and able to proceed through the reductive half of the catalytic cycle, but the enzyme no longer reacted with oxygen. The effect of Na 1 and Li 1 was source-specific for pig kidney and bovine kidney amine oxidases, while the enzymes from bovine serum or plants were not inactivated, consistent with a mechanism dependent on small structural differences. A model for irreversible inactivation is proposed in which the cofactor is co-ordinated directly to copper, in analogy with the inactivation reported for Escherichia coli amine oxidase under crystal growth conditions. Keywords: pig kidney; amine oxidase; copper; 6-hydroxydopa; cations. , and from Euphorbia characias latex [12]. All these amine oxidases are homodimers in which each subunit (molecular mass 70±90 kDa) contains one tightly bound Cu II ion and one 6-hydroxydopa quinone (TPQ) [13] as prosthetic groups. TPQ is derived from the oxidation in a post-translational event of a tyrosinyl residue in the amino-acid sequence [14±17]. The crystal structure of some amine oxidases shows that the copper atom is co-ordinated by three histidine side chains and by two water molecules [18±21], and that TPQ is approximately 4±5 A Ê away from the copper ion. A hydrophobic cavity has been described near the O-2 position of the reduced cofactor and has been proposed to be the dioxygen binding site [18,22].Amine oxidases catalyze the oxidation of primary amines, including mono, di and polyamines, with the formation of the corresponding aldehyde, ammonia and hydrogen peroxide. The catalytic mechanism can be divided into two half-reactions, namely the enzyme reduction by substrate (Eqn 1), followed by re-oxidation by molecular oxygen (Eqn 2):The catalytic mechanism of copper±amine oxidases is detailed in Scheme 1, [23]. While detailed studies of the reductive half-reaction (Eqn 1) (Scheme 1, a±e) have been reported [24±28], little is known about the oxidative half-reaction (Eqn 2). In plant amine oxidases, the Cu IIaminoresorcinol (e) can undergo a one-electron transfer to the Cu II center, generating an EPR-detectable Cu I -semiquinolamine free radical species (f), characterized by absorption bands at 464, 434, and 360 nm [27±33]. In pig kidney amine oxidase (PKAO), th...
Copper amine oxidase from lentil seedlings was shown to be able to catalyze the oxidative deamination of the indoleamines tryptamine, 5-hydroxytryptamine, and 5-methoxytryptamine. These compounds showed saturation kinetics with K m values as normal substrates, but their oxidation led to irreversible loss of enzyme activity suggesting a covalent interaction with the enzyme, most probably through its cofactor 6-hydroxydopa (2,4,5-trihydroxyphenylalanine). These indoleamines acted as irreversible inhibitors of the enzyme only in the absence of oxygen but they brought about changes in the electronic spectra of the enzyme both in aerobiosis and in anaerobiosis. This study reports on the mechanism by which these compounds inhibit lentil amine oxidase which involves first the oxidation of indoleamines bound to 6-hydroxydopa followed by the formation of an irreversible covalent derivative. The same inhibitory mechanism could possibly lead to inactivation of mammalian amine oxidases involved in serotonin neurotransmitter metabolism in conditions of ischemia or hypoxia.
In this review, inhibitors of plant copper amine oxidases from Lens esculenta seedlings, Pisum sativum seedlings, and Euphorbia characias latex are described. Reversible competitive inhibitors and non-competitive inhibitors, irreversible active-site directed inhibitors and mechanism-based inactivators are reviewed in regard to their mechanisms of action.
The oxidation of L-ornithine and L-arginine catalyzed by lentil (Lens esculenta) seedling copper-amine oxidase has been investigated by polarographic techniques, optical spectroscopy, and capillary electrophoresis. Both L-ornithine and L-arginine were found to be poor substrates for lentil amine oxidase. L-Ornithine was oxidized to glutamate-5-semialdehyde and ammonia, in similar manner as usual substrates. Glutamate-5-semialdehyde spontaneously cyclizes to delta1-pyrroline-5-carboxylic acid. Arginine is oxidized by an unusual mechanism yielding glutamate-5-semialdehyde, ammonia, and urea as reaction products.
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