6 Hydroxydopa

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Floris G - One of the best experts on this subject based on the ideXlab platform.

  • An important lysine residue in copper⁄quinone-containing amine oxidases
    'Wiley', 2007
    Co-Authors: Mura A, Floris G, Padiglia Alessandra, Anedda R, Pintus Francesca, Casu Mariano, Medda Rosaria
    Abstract:

    The interaction of xenon with copper/6-Hydroxydopa (2,4,5-trihydroxyphenethylamine) quinone (TPQ) amine oxidases from the plant pulses lentil (Lens esculenta) and pea (Pisum sativum) (seedlings), the perennial Mediterranean shrub Euphorbia characias (latex), and the mammals cattle (serum) and pigs (kidney), were investigated by NMR and optical spectroscopy of the aqueous solutions of the enzymes. (129)Xe chemical shift provided evidence of xenon binding to one or more cavities of all these enzymes, and optical spectroscopy showed that under 10 atm of xenon gas, and in the absence of a substrate, the plant enzyme cofactor (TPQ), is converted into its reduced semiquinolamine radical. The kinetic parameters of the analyzed plant amine oxidases showed that the k(c) value of the xenon-treated enzymes was reduced by 40%. Moreover, whereas the measured K(m) value for oxygen and for the aromatic monoamine benzylamine was shown to be unchanged, the K(m) value for the diamine putrescine increased remarkably after the addition of xenon. Under the same experimental conditions, the TPQ of bovine serum amine oxidase maintained its oxidized form, whereas in pig kidney, the reduced aminoquinol species was formed without the radical species. Moreover the k(c) value of the xenon-treated pig enzyme in the presence of both benzylamine and cadaverine was shown to be dramatically reduced. It is proposed that the lysine residue at the active site of amine oxidase could be involved both in the formation of the reduced TPQ and in controlling catalytic activity

  • Nitric oxide covalently labels a 6-Hydroxydopa-derived free radical intermediate in the catalytic cycle of copper/quinone-containing amine oxidase from lentil seedlings
    Walter de Gruyter and Company, 2005
    Co-Authors: Longu S, Padiglia A, Jz Pedersen, Finazzi Agro A, Mura A, Maccioni P, Floris G, Medda R
    Abstract:

    The reaction of NO-derivatized polyamines called 'NONOates' with an amine oxidase from lentil seedlings was studied. 3,3-Bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (DETA-NONOate) and 3,3'-(hydroxynitrosohydrazino)bis-1-propanamine (DPTA-NONOate) were found to be irreversible inactivators of the lentil enzyme. The spectrum of the protein was strongly affected in the course of reaction with both compounds, leading to the formation of a covalent adduct with a stable band at 334 nm. The corresponding amine compounds diethylentriamine (DETA) and norspermidine (DPTA) were substrates of the lentil enzyme that did not lead to enzyme inactivation. Diethylamine-NONOate, not containing amino groups, was found to be an irreversible inactivator of the amine oxidase only in the presence of a substrate. Since all NONOates spontaneously decompose in solution with release of NO, it seems as if the latter is responsible for the enzyme inhibition. The insensitivity of the native enzyme to NO suggested that this compound was unreactive toward both the cofactors, 6-Hydroxydopa quinone (TPQ) and Cu(II), and thus a model for the irreversible inactivation could involve the attack by NO of the Cu(I)-semiquinolamine radical catalytic intermediate

  • Copper/Topaquinone–containing amine oxidase from lentil seedlings and bovine plasma: catalytic mechanism and energetic domains
    2004
    Co-Authors: Medda Rosaria, Longu S, Floris G, Agostinelli E, Dalla Vedova L, Pedersen J. Z, Moosavi Movahedi A. A, Padiglia Alessandra
    Abstract:

    In this review the characteristics of the prosthetic group and the role of copper in amine oxidase purified from lentil seedlings are compared with the corresponding features of the amine oxidase isolated from bovine serum. Although both enzymes contain the same organic cofactor, the 6-Hydroxydopa (2,4,5-trihydroxyphenethylamine) quinone, the catalytic cycle of lentil seedling amine oxidase operates through a Cu(I)-free-radical intermediate of the cofactor, whereas in bovine serum enzyme the radical form was not observed. The role of the metal in the catalytic mechanism of the two enzymes is also discussed. Moreover, the energetic domains and the effect of the temperature on activity, for both enzymes, are examined using differential scanning calorimetry

  • Effect of metal substitution in copper amine oxidase from lentil seedlings
    'Springer Science and Business Media LLC', 1999
    Co-Authors: Padiglia A, Medda R, Lorrai A, Murgia B, Floris G
    Abstract:

    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, Go-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, Go-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 Go-substituted enzyme is the organic cofactor 6-Hydroxydopa quinone reactive and the enzyme catalytically competent, although less efficient. The Go-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

  • Characterization of Euphorbia characias latex amine oxidase.
    'American Society of Plant Biologists (ASPB)', 1998
    Co-Authors: Padiglia A, Finazzi Agro A, Medda R, Lorrai A, Murgia B, Floris G
    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 spectros-copy under both aerobic and anaerobic conditions. The extinction coefficients at 278 and 490 nm were determined as 3.78 x 105 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 hexapeplide present in most 6-Hydroxydopa quinone-containing amine oxidases. This hexapeptide contains the tyrosinyI residue that can be modified into the cofactor 6-Hydroxydopa quinone

Medda R - One of the best experts on this subject based on the ideXlab platform.

  • Nitric oxide covalently labels a 6-Hydroxydopa-derived free radical intermediate in the catalytic cycle of copper/quinone-containing amine oxidase from lentil seedlings
    Walter de Gruyter and Company, 2005
    Co-Authors: Longu S, Padiglia A, Jz Pedersen, Finazzi Agro A, Mura A, Maccioni P, Floris G, Medda R
    Abstract:

    The reaction of NO-derivatized polyamines called 'NONOates' with an amine oxidase from lentil seedlings was studied. 3,3-Bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (DETA-NONOate) and 3,3'-(hydroxynitrosohydrazino)bis-1-propanamine (DPTA-NONOate) were found to be irreversible inactivators of the lentil enzyme. The spectrum of the protein was strongly affected in the course of reaction with both compounds, leading to the formation of a covalent adduct with a stable band at 334 nm. The corresponding amine compounds diethylentriamine (DETA) and norspermidine (DPTA) were substrates of the lentil enzyme that did not lead to enzyme inactivation. Diethylamine-NONOate, not containing amino groups, was found to be an irreversible inactivator of the amine oxidase only in the presence of a substrate. Since all NONOates spontaneously decompose in solution with release of NO, it seems as if the latter is responsible for the enzyme inhibition. The insensitivity of the native enzyme to NO suggested that this compound was unreactive toward both the cofactors, 6-Hydroxydopa quinone (TPQ) and Cu(II), and thus a model for the irreversible inactivation could involve the attack by NO of the Cu(I)-semiquinolamine radical catalytic intermediate

  • Effect of metal substitution in copper amine oxidase from lentil seedlings
    'Springer Science and Business Media LLC', 1999
    Co-Authors: Padiglia A, Medda R, Lorrai A, Murgia B, Floris G
    Abstract:

    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, Go-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, Go-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 Go-substituted enzyme is the organic cofactor 6-Hydroxydopa quinone reactive and the enzyme catalytically competent, although less efficient. The Go-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

  • Characterization of Euphorbia characias latex amine oxidase.
    'American Society of Plant Biologists (ASPB)', 1998
    Co-Authors: Padiglia A, Finazzi Agro A, Medda R, Lorrai A, Murgia B, Floris G
    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 spectros-copy under both aerobic and anaerobic conditions. The extinction coefficients at 278 and 490 nm were determined as 3.78 x 105 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 hexapeplide present in most 6-Hydroxydopa quinone-containing amine oxidases. This hexapeptide contains the tyrosinyI residue that can be modified into the cofactor 6-Hydroxydopa quinone

  • Tryptamine as substrate and inhibitor of lentil seedling copper amine oxidase
    1997
    Co-Authors: Medda R, Padiglia A, Pedersen J, Finazzi Agrò A, Lorrai A, Floris G
    Abstract:

    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 Km 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

  • Inhibition of copper amine oxidase by haloamines: A killer product mechanism
    1997
    Co-Authors: Medda R, Padiglia A, Finazzi Agro A, Pedersen J, Rotilio G, Floris G
    Abstract:

    The observation that the alkylamines 2-Br-ethylamine and 2-C1-ethylamine and 1,2-diaminoethane, the shortest diamine, are irreversible inhibitors of several copper amine oxidases led to the investigation of the mechanism by which these compounds react with the highly active amine oxidase from lentil seedlings. 1,2-Diaminoethane, 2-Br-ethylamine, and 2-C1-ethylamine were found to be both poor substrates and irreversible inhibitors of lentil amine oxidase; inactivation took place in both the presence and absence of oxygen. All three compounds strongly affected the spectrum of the enzyme, leading to the formation of a stable band at 336 nm both in anaerobiosis and in aerobiosis, consistent with an interaction with the enzyme cofactor 6-Hydroxydopa. On the contrary, the corresponding propylamine compounds 1,3-diaminopropane, 3-Br-propylamine, and 3-C1-propylamine were reversible inhibitors of lentil amine oxidase. Inhibition was shown to be due to the aldehyde oxidation products rather than the short chain amines themselves; a reaction mechanism is presented which involves attack of the aldehyde on the 6-Hydroxydopa-derived free radical catalytic intermediate. With 1,2-diaminoethane, 2-Br-ethylamine, and 2-C1-ethylamine, the complex produced will form a stable 6-membered ring, causing irreversible inhibition of the enzyme

Padiglia A - One of the best experts on this subject based on the ideXlab platform.

  • Nitric oxide covalently labels a 6-Hydroxydopa-derived free radical intermediate in the catalytic cycle of copper/quinone-containing amine oxidase from lentil seedlings
    Walter de Gruyter and Company, 2005
    Co-Authors: Longu S, Padiglia A, Jz Pedersen, Finazzi Agro A, Mura A, Maccioni P, Floris G, Medda R
    Abstract:

    The reaction of NO-derivatized polyamines called 'NONOates' with an amine oxidase from lentil seedlings was studied. 3,3-Bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (DETA-NONOate) and 3,3'-(hydroxynitrosohydrazino)bis-1-propanamine (DPTA-NONOate) were found to be irreversible inactivators of the lentil enzyme. The spectrum of the protein was strongly affected in the course of reaction with both compounds, leading to the formation of a covalent adduct with a stable band at 334 nm. The corresponding amine compounds diethylentriamine (DETA) and norspermidine (DPTA) were substrates of the lentil enzyme that did not lead to enzyme inactivation. Diethylamine-NONOate, not containing amino groups, was found to be an irreversible inactivator of the amine oxidase only in the presence of a substrate. Since all NONOates spontaneously decompose in solution with release of NO, it seems as if the latter is responsible for the enzyme inhibition. The insensitivity of the native enzyme to NO suggested that this compound was unreactive toward both the cofactors, 6-Hydroxydopa quinone (TPQ) and Cu(II), and thus a model for the irreversible inactivation could involve the attack by NO of the Cu(I)-semiquinolamine radical catalytic intermediate

  • Effect of metal substitution in copper amine oxidase from lentil seedlings
    'Springer Science and Business Media LLC', 1999
    Co-Authors: Padiglia A, Medda R, Lorrai A, Murgia B, Floris G
    Abstract:

    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, Go-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, Go-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 Go-substituted enzyme is the organic cofactor 6-Hydroxydopa quinone reactive and the enzyme catalytically competent, although less efficient. The Go-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

  • Characterization of Euphorbia characias latex amine oxidase.
    'American Society of Plant Biologists (ASPB)', 1998
    Co-Authors: Padiglia A, Finazzi Agro A, Medda R, Lorrai A, Murgia B, Floris G
    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 spectros-copy under both aerobic and anaerobic conditions. The extinction coefficients at 278 and 490 nm were determined as 3.78 x 105 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 hexapeplide present in most 6-Hydroxydopa quinone-containing amine oxidases. This hexapeptide contains the tyrosinyI residue that can be modified into the cofactor 6-Hydroxydopa quinone

  • Tryptamine as substrate and inhibitor of lentil seedling copper amine oxidase
    1997
    Co-Authors: Medda R, Padiglia A, Pedersen J, Finazzi Agrò A, Lorrai A, Floris G
    Abstract:

    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 Km 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

  • Inhibition of copper amine oxidase by haloamines: A killer product mechanism
    1997
    Co-Authors: Medda R, Padiglia A, Finazzi Agro A, Pedersen J, Rotilio G, Floris G
    Abstract:

    The observation that the alkylamines 2-Br-ethylamine and 2-C1-ethylamine and 1,2-diaminoethane, the shortest diamine, are irreversible inhibitors of several copper amine oxidases led to the investigation of the mechanism by which these compounds react with the highly active amine oxidase from lentil seedlings. 1,2-Diaminoethane, 2-Br-ethylamine, and 2-C1-ethylamine were found to be both poor substrates and irreversible inhibitors of lentil amine oxidase; inactivation took place in both the presence and absence of oxygen. All three compounds strongly affected the spectrum of the enzyme, leading to the formation of a stable band at 336 nm both in anaerobiosis and in aerobiosis, consistent with an interaction with the enzyme cofactor 6-Hydroxydopa. On the contrary, the corresponding propylamine compounds 1,3-diaminopropane, 3-Br-propylamine, and 3-C1-propylamine were reversible inhibitors of lentil amine oxidase. Inhibition was shown to be due to the aldehyde oxidation products rather than the short chain amines themselves; a reaction mechanism is presented which involves attack of the aldehyde on the 6-Hydroxydopa-derived free radical catalytic intermediate. With 1,2-diaminoethane, 2-Br-ethylamine, and 2-C1-ethylamine, the complex produced will form a stable 6-membered ring, causing irreversible inhibition of the enzyme

Alessandra Padiglia - One of the best experts on this subject based on the ideXlab platform.

  • Copper/topaquinone-containing amine oxidase from lentil seedlings and bovine plasma: Catalytic mechanism and energetic domains
    'Springer Science and Business Media LLC', 2004
    Co-Authors: R. Medda, Jz Pedersen, S. Longu, Enzo Agostinelli, Dalla L. Vedova, G. Floris, A.a. Moosavi-movahedi, Alessandra Padiglia
    Abstract:

    In this review the characteristics of the prosthetic group and the role of copper in amine oxidase purified from lentil seedlings are compared with the corresponding features of the amine oxidase isolated from bovine serum. Although both enzymes contain the same organic cofactor, the 6-Hydroxydopa ( 2,4,5-trihydroxyphenethylamine) quinone, the catalytic cycle of lentil seedling amine oxidase operates through a Cu(I)-free-radical intermediate of the cofactor, whereas in bovine serum enzyme the radical form was not observed. The role of the metal in the catalytic mechanism of the two enzymes is also discussed. Moreover, the energetic domains and the effect of the temperature on activity, for both enzymes, are examined using differential scanning calorimetry

  • Oxidation of benzylamine Br-derivatives by lentil seedling copper-amine oxidase
    Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology, 2000
    Co-Authors: Rosaria Medda, Alessandra Padiglia, Anita Lorrai, Barbara Murgia, Giovanni Floris
    Abstract:

    ABSTRACT Copper amine oxidase was shown to be able to catalyse the oxidative deamination of 2-, 3- and 4-Br-derivatives of benzylamine to the corresponding aldehydes, that all absorb at 250 nm. This change in the absorption spectrum made it possible to follow the enzyme reaction. 2-Br-benzylamine, 3-Br-benzylamine, and 4-Br-benzylamine showed Km values similar to benzylamine, but 3-Br-benzylamine showed a slower kc, which allows it to be a catalytically more efficient substrate. Under anaerobic conditions the native enzyme oxidised 1 equivalent of all Br-derivatives and released 1 equivalent of aldehyde per enzyme subunit. These findings demonstrate that, in anaerobic conditions, the enzyme can oxidise substrates with a single incomplete turnover. The possible involvement of the cofactor 6-Hydroxydopa quinone and of a negatively charged residue in the oxidation of Br-benzylamines is discussed.

Pedersen J - One of the best experts on this subject based on the ideXlab platform.

  • Tryptamine as substrate and inhibitor of lentil seedling copper amine oxidase
    1997
    Co-Authors: Medda R, Padiglia A, Pedersen J, Finazzi Agrò A, Lorrai A, Floris G
    Abstract:

    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 Km 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

  • Inhibition of copper amine oxidase by haloamines: A killer product mechanism
    1997
    Co-Authors: Medda R, Padiglia A, Finazzi Agro A, Pedersen J, Rotilio G, Floris G
    Abstract:

    The observation that the alkylamines 2-Br-ethylamine and 2-C1-ethylamine and 1,2-diaminoethane, the shortest diamine, are irreversible inhibitors of several copper amine oxidases led to the investigation of the mechanism by which these compounds react with the highly active amine oxidase from lentil seedlings. 1,2-Diaminoethane, 2-Br-ethylamine, and 2-C1-ethylamine were found to be both poor substrates and irreversible inhibitors of lentil amine oxidase; inactivation took place in both the presence and absence of oxygen. All three compounds strongly affected the spectrum of the enzyme, leading to the formation of a stable band at 336 nm both in anaerobiosis and in aerobiosis, consistent with an interaction with the enzyme cofactor 6-Hydroxydopa. On the contrary, the corresponding propylamine compounds 1,3-diaminopropane, 3-Br-propylamine, and 3-C1-propylamine were reversible inhibitors of lentil amine oxidase. Inhibition was shown to be due to the aldehyde oxidation products rather than the short chain amines themselves; a reaction mechanism is presented which involves attack of the aldehyde on the 6-Hydroxydopa-derived free radical catalytic intermediate. With 1,2-diaminoethane, 2-Br-ethylamine, and 2-C1-ethylamine, the complex produced will form a stable 6-membered ring, causing irreversible inhibition of the enzyme

  • The reaction mechanism of copper amine oxidase: detection of intermediates by the use of substrates and inhibitors
    1995
    Co-Authors: Medda R, Padiglia A, Pedersen J, Rotilio G, Finazzi Agrò A, Floris G
    Abstract:

    Intermediate states in the catalytic mechanism of lentil copper amine oxidase have been investigated by ESR and optical spectroscopy. Using highly purified apo- and holoenzyme in combination with a poor substrate and a range of inhibitors, under both aerobic and anaerobic conditions, the single steps of the reaction mechanism can be slowed down or 'frozen' completely. In this way, a sequence of six intermediate species in the catalytic cycle has been established. Oxidative deamination of p-(dimethylamino)benzylamine is 5 x 10(5) times slower than for putrescine; the rate-limiting step is shown to be release of the aldehyde product. This process is not affected in the apoenzyme, but subsequent intramolecular electron transfer to form the characteristic free radical intermediate is completely blocked, and the apoenzyme is trapped as an aminoresorcinol species. Classic hydrazine and hydrazide inhibitors bind to the 6-Hydroxydopa cofactor in the same way as active substrates, but rearrangements lead to formation of stable intermediate adducts at the step preceding release of aldehyde. The semicarbazide-6-Hydroxydopa adduct is shown to bind simultaneously to Cu(II), providing the first direct evidence for localization of 6-Hydroxydopa close to the copper site

  • A substrate-cofactor free radical intermediate in the reaction mechanism of copper amine oxidase
    1992
    Co-Authors: Pedersen J, Finazzi Agrò A, El-sherbini S, Rotilio G
    Abstract:

    Reduction of copper amine oxidase with substrate led to the appearance of a free radical which can be detected in anaerobiosis by ESR and optical spectroscopy. The origin of this radical was examined through studies of the semiquinones of 6-Hydroxydopamine, an analogue of the recently identified cofactor 6-Hydroxydopa. The ESR spectrum of the 6-Hydroxydopamine radical was too narrow to account for the enzyme radical signal; however, after spontaneous reaction with primary amines the hyperfine splittings and spectral width obtained by modulation broadening became very similar to those observed for the oxidase radical species. This effect was ascribed to covalent binding of a nitrogen atom directly to the aromatic ring structure, suggesting that the amine oxidase radical is an amino-6-Hydroxydopa semiquinone. Identical ESR spectra were obtained using the amines putrescine, cadaverine, p-[(dimethylamino)methyl]benzylamine, and ethylenediamine; these oxidase substrates gave identical enzyme radical spectra as well. The interaction between cofactor and substrate was proved unambiguously by the technique of isotopic labeling: addition of [15N2]ethylenediamine instead of the normal 14N-labeled compound changed the ESR spectra of both the enzyme radical and its 6-Hydroxydopamine counterpart. The results were confirmed by optical spectroscopy measurements; 6-Hydroxydopamine and oxidized 6-Hydroxydopamine gave spectra identical to those of reduced and oxidized amine oxidase, respectively. The 6-Hydroxydopamine radical showed a sharp peak at 440 nm; upon addition of amines the maximum shifted to 460 nm, as found for the enzyme. It is proposed that copper amine oxidase represents the first example of a mixed substrate-cofactor radical within the family of tyrosine radical enzymes