The Reductive and Oxidative Half‐Reactions and the Role of Copper Ions in Plant and Mammalian Copper−Amine Oxidases

Padiglia, Alessandra; Medda, Rosaria; Bellelli, Andrea; Agostinelli, Enzo; Morpurgo, Laura; Mondovı̀, Bruno; Agró, Alessandro Finazzi; Floris, Giovanni

doi:10.1002/1099-0682(20011)2001:1<35::aid-ejic35>3.0.co;2-#

Cited by 34 publications

(14 citation statements)

References 74 publications

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“…However, an obligatory role for Cu(I) has not been established, and a viable mechanistic alternative that does not invoke a change in the oxidation state of copper has been proposed [79,80]. Copper amine oxidases from different sources have been suggested to use distinctly different mechanisms for reoxidation [81]. We anticipate that the work presented herein will facilitate experimental design to probe the molecular details of TPQ reoxidation and the concomitant reduction of dioxygen in the human enzyme.…”

Section: Discussionmentioning

confidence: 88%

Human kidney diamine oxidase: heterologous expression, purification, and characterization

2002

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Human kidney diamine oxidase has been overexpressed as a secreted enzyme under the control of a metallothionein promoter in Drosophila S2 cell culture. This represents the first heterologous overexpression and purification of a catalytically active, recombinant mammalian copper-containing amine oxidase. A rapid and highly efficient purification protocol using chromatography on heparin affinity, hydroxyapatite, and gel filtration media allows for the recovery of large quantities of the recombinant enzyme, which is judged to be greater than 98% homogenous by SDS/PAGE. The availability of large quantities of highly purified enzyme makes it now possible to investigate the spectroscopic, mechanistic, functional, and structural properties of this human enzyme at the molecular level. Visible absorption, circular dichroism, electron paramagnetic resonance, and resonance Raman spectroscopic results are presented. The recombinant enzyme contains the cofactors 2,4,5-trihydroxyphenylalaninequinone and copper at stoichiometries of up to 1.1 and 1.5 mol per mol homodimer, respectively. In addition, tightly bound and stoichiometric calcium ions were identified and proposed to occupy a second metal-binding site. The apparent molecular weight of the recombinant protein, determined by analytical ultracentrifugation, suggests 20-26% glycosylation by weight. Detailed kinetic studies indicate the preferred substrates (k(cat)/K(M)) of human diamine oxidase are, in order, histamine, 1-methylhistamine, and putrescine, with K(M) values of 2.8, 3.4, and 20 microM, respectively. These results, demonstrating the substrate preference for histamine and 1-methylhistamine, were unanticipated given the available literature. The pH dependence of k(cat) for putrescine oxidation gives two apparent p K(a) values at 6.0 and 8.2. Tissue-specific expression of the human diamine oxidase gene was investigated using an mRNA array. The relevance of this work to earlier work and the suggested physiological roles of the human enzyme are discussed.

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Section: Discussionmentioning

confidence: 88%

Human kidney diamine oxidase: heterologous expression, purification, and characterization

2002

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“…They catalyze the oxidative deamination of primary amines to aldehydes with a pingpong mechanism consisting of a transamination [2], followed by the transfer of two electrons to molecular oxygen which is reduced to H 2 O 2 (Eqs. (1) and (2)).…”

Section: Introductionmentioning

confidence: 99%

“…The structural similarity does not prevent amine oxidases of various sources from displaying different reactivity with substrates and inhibitors [2,18]. An example is provided by LSAO and the amine oxidase from bovine serum (BSAO).…”

Section: Introductionmentioning

confidence: 99%

Substrate specificity of copper-containing plant amine oxidases

Pietrangeli

Federico

Mondovı̀

et al. 2007

Journal of Inorganic Biochemistry

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“…On the basis of the results of kinetic and spectroscopic studies, [19][20][21][22][23][24][25] as well as structural and functional studies using site-directed mutant variants, [26][27][28] the catalytic mechanism is now quite well established. [29][30][31][32] The amine oxidation occurs via a ping pong mechanism: in the first step, TPQ is reduced to an aminoquinol form and the amine substrate is oxidized to aldehyde; in the second step, the organic cofactor is re-oxidized at the expense of molecular oxygen, which is reduced to hydrogen peroxide. 29 However, some fundamental questions, such as structural aspects of substrate specificity, the catalytic role of the copper ion and possible cooperativity/communication between the subunits, 29,[33][34][35] are still unresolved.…”

Section: Introductionmentioning

confidence: 99%