RESUMO
The steady-state kinetic parameters of the amine oxidases purified from Lathyrus cicera (LCAO) and Pisum sativum (PSAO) seedling were measured on a series of common substrates, previously tested on bovine serum amine oxidase (BSAO). LCAO, as PSAO, was substantially more reactive than BSAO with aliphatic diamines and histamine. The k(cat) and k(cat)/Km for putrescine were four and six order of magnitude higher, respectively. Differences were smaller with some aromatic monoamines. The plot of k(cat) versus hydrogen ions concentration produced bell-shaped curves, the maximum of which was substrate dependent, shifting from neutral pH with putrescine to alkaline pH with phenylethylamine and benzylamine. The latter substrates made the site more hydrophobic and increased the pK(a) of both enzyme-substrate and enzyme-product adducts. The plot of k(cat)/Km versus hydrogen ion concentration produced approximately parallel bell-shaped curves. Similar pK(a) couples were obtained from the latter curves, in agreement with the assignment as free enzyme and free substrate pK(a). The limited pH dependence of kinetic parameters suggests a predominance of hydrophobic interactions.
Assuntos
Amina Oxidase (contendo Cobre)/metabolismo , Proteínas de Plantas/metabolismo , Amina Oxidase (contendo Cobre)/química , Aminas/química , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Estrutura Molecular , Proteínas de Plantas/química , Especificidade por SubstratoRESUMO
This short review is mostly concerned with the work carried out in Rome on the copper amine oxidase from bovine serum (BSAO). The first target was the copper oxidation state and its relationship with the organic cofactor. It was found that copper is not reduced on reaction with amines under anaerobic conditions or along the catalytic cycle and that it is not within bonding distance of the quinone cofactor. The copper stability in the oxidised state was supported by BSAO ability to oxidise benzylhydrazine, a slow substrate, in the presence of N,N-diethyldithiocarbamate (DDC) and by the substantial catalytic activity of Co(2+)-substituted BSAO. Parallel work established that only one subunit of the dimeric enzyme readily binds reagents of the carbonyl group. Flexible hydrazides with a long aromatic tail were found to be highly specific inhibitors, suggesting the presence of an extended hydrophobic region at the catalytic site. A study by stopped-flow transient spectroscopy and steady state kinetics led to the formulation of a simplified, yet complete and consistent, catalytic mechanism for BSAO that was compared with that available for lentil seedling amine oxidase (LSAO). As in other copper amine oxidases, BSAO is inactivated by H(2)O(2) produced in the catalytic reaction, while the cofactor is stabilised in its reduced state. A conserved tyrosine hydrogen-bonded to the cofactor might be oxidised.
Assuntos
Amina Oxidase (contendo Cobre)/metabolismo , Bactérias/enzimologia , Di-Hidroxifenilalanina/análogos & derivados , Di-Hidroxifenilalanina/metabolismo , Plantas/enzimologia , Amina Oxidase (contendo Cobre)/química , Catálise , Peróxido de Hidrogênio/farmacologia , Oxirredução , Subunidades ProteicasRESUMO
Two contrasting topics are examined in this account: the protective actions of amine oxidases (AOs) resulting from the elimination and/or modulation of the levels of polyamines and some biogenic amines, such as histamine, in anaphylactic shock and the cell damaging effect of AOs catabolic products. Other functions of the plasma copper-containing amine oxidase are considered; namely the modification of some proteins by oxidation of their free amino groups, the auto-regulation of the catalytic activity of AOs, the protective effect against free radicals, and the regulation of K(+)-channels.
RESUMO
The presteady-state and steady-state kinetics of bovine serum amine oxidase (BSAO) were analyzed by stopped-flow transient spectroscopy. A simplified model of the catalytic cycle was found to describe the experimental data and the rate constants of the individual steps were used to calculate Michaelis parameters that agree with the direct determinations. In spite of many studies on selected reactions from the catalytic cycle, this is amongst the first efforts to provide a comprehensive kinetic description of the reactions of BSAO, whose results can be compared with the steady-state parameters. The reoxidation reaction by dioxygen is more complex than previously thought, in agreement with a recent report [Su, Q. & Klinman, J.P. (1998) Biochemistry 37, 12513-12525], and occurs in at least two steps whose rate constants, previously undetermined, have been measured. The reaction of the oxidized enzyme with the amine substrate is poorly determined in this type of experiment, thus irreversible combination with aromatic hydrazine inhibitors was used as a model system, demonstrating that the mechanism and rate constants of their reaction is fully compatible with an accurate description of the catalytic cycle with the physiological substrate. These results constitute a simplified, yet complete and consistent, description of the catalytic cycle and offer an interesting comparison with those obtained on plant amine oxidases; two steps of the catalytic cycle are significantly slower in BSAO than in pea seedling or lentil seedling amine oxidases, namely the reoxidation and the trans-iminative proton abstraction occurring in the enzyme-substrate complex. The former difference is rationalized as being due to the low to zero concentration of the semiquinolamine-radical intermediate, while the latter is less easily interpreted.
Assuntos
Amina Oxidase (contendo Cobre)/metabolismo , Amina Oxidase (contendo Cobre)/antagonistas & inibidores , Animais , Catálise , Bovinos , Coenzimas/metabolismo , Di-Hidroxifenilalanina/análogos & derivados , Di-Hidroxifenilalanina/metabolismo , Inibidores Enzimáticos/farmacologia , Hidrazinas/farmacologia , Cinética , Oxirredução , Oxigênio/metabolismo , Fenil-Hidrazinas/farmacologiaRESUMO
Bovine serum amine oxidase (BSAO), reduced by excess amine under limited turnover conditions, was over 80% inactivated by H(2)O(2) upon oxygen exhaustion. The UV-Vis spectrum and the reduced reactivity with carbonyl reagents showed that the cofactor topaquinone (TPQ) was stabilized in reduced form. The protein large M(r) (170 kDa) prevented the identification of modified residues by amino acid analyses. Minor changes of the Cu(2+) EPR signal and the formation of a radical at g = 2.001, with intensity a few percent of that of the Cu(2+) signal, unaffected by a temperature increase, suggest that Cu(2+)-bound histidines were not oxidized and the radical was not the Cu(+)-semiquinolamine in equilibrium with Cu(2+)-aminoquinol. It may derive from the modification of a conserved residue in proximity of the active site, possibly the tyrosine at hydrogen-bonding distance of TPQ C-4 ionized hydroxyl. The inactivation reaction appears to be a general feature of copper-containing amine oxidases. It may be part of an autoregulatory process in vivo, possibly relevant to cell adhesion and redox signaling.
Assuntos
Amina Oxidase (contendo Cobre)/sangue , Peróxido de Hidrogênio/farmacologia , Amina Oxidase (contendo Cobre)/química , Amina Oxidase (contendo Cobre)/isolamento & purificação , Animais , Proteínas Sanguíneas/metabolismo , Bovinos , Espectroscopia de Ressonância de Spin Eletrônica , Cinética , Peso Molecular , Espermina/farmacologiaRESUMO
Bovine serum amine oxidase (BSAO) reacts with 2-hydrazinopyridine, which binds the organic co-factor 2,4,5-trihydroxyphenylalanine quinone, forming a band at 435 nm. The band shifts to 526 nm around 60 degrees C, to 415 nm upon denaturation, but only shifts to 429 nm upon Cu2+ depletion. Its wavelength and intensity suggest that the adduct has the azo conformation, whilst the same adduct of crystalline Escherichia coli amine oxidase (ECAO) shows the hydrazone conformation in the X-ray structure. The steady state kinetics of aminomethyl- and aminoethylpyridines confirm that the formation of the product Schiff base, analogous to the azo form of the 2-hydrazinopyridine adduct, is not hindered in solution. The structural stability of the adduct in the absence of Cu2+ is taken to imply hydrogen bonding of the pyridyl nitrogen to a conserved aspartate, as in the ECAO adduct. Thus the ECAO adduct provides a good model for a transient intermediate leading to formation of the BSAO azo adduct. On the basis of this model and of the catalytic competence of Co(2+)-substituted BSAO, confirmed by the present data, a catalytic reaction scheme is proposed.
Assuntos
Amina Oxidase (contendo Cobre)/química , Amina Oxidase (contendo Cobre)/metabolismo , Metais/metabolismo , Amina Oxidase (contendo Cobre)/antagonistas & inibidores , Animais , Bovinos , Cobalto/química , Cobalto/metabolismo , Cobre/química , Cobre/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Cinética , Piridonas/química , Piridonas/farmacologiaRESUMO
Haemoglobin has been previously identified as responsible for the decreased enzymatic activity of copper bovine serum amine oxidase (BSAO) in suspensions of human or bovine hemolyzed erythrocytes [Marcocci, L., Pietrangeli, P., Befani, O., Mavelli, I., & Mondovi', B. (1991b) Life Chem. Report, 9, 171-177]. This is confirmed by present results on bovine methaemoglobin. Bovine globin and horse skeletal muscle mioglobin showed a similar inhibiting ability, but neither bovine serum albumin nor cytochrome c inhibited BSAO activity under the same experimental conditions. The inhibitory effect of bovine haemoglobin was dependent on pH only at high buffer ionic strength. It was highest in physiological conditions (PBS) where haemoglobin acted as a reversible non competitive inhibitor of BSAO activity, with apparent Ki of 0.5 mM at 37 degrees C. The inhibition was unaffected by partial BSAO deglycosylation (40% of glucidic residues removed) but decreased when haemoglobin lysine groups were derivatised using citraconic anhydride. A possible molecular mechanism underlying the inhibitory effect is discussed.
Assuntos
Amina Oxidase (contendo Cobre) , Metemoglobina/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/antagonistas & inibidores , Animais , Benzilaminas/metabolismo , Proteínas Sanguíneas/antagonistas & inibidores , Bovinos , Humanos , Cinética , Peso Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/sangue , Especificidade por SubstratoRESUMO
The unmediated electrochemistry of two large Cu-containing proteins, ascorbate oxidase and laccase, was investigated by direct-current cyclic voltammetry. Rapid heterogeneous electron transfer was achieved in the absence of promoters or mediators by trapping a small amount of protein within a solid, electrochemically inert, tributylmethyl phosphonium chloride membrane coating a gold electrode. The problems typical of proteins in solution, such as adsorption on the electrode surface, were avoided by this procedure. In anaerobic conditions, the cyclic voltammograms, run at a scan rate of up to 200 mV/s, showed the electron transfer process to be quasi-reversible and diffusion-controlled. The pH-dependent redox potentials (+360 mV and +400 mV against a normal hydrogen electrode at pH7.0 for ascorbate oxidase and laccase respectively and +390 mV and +410 mV at pH5.5) were similar to those of the free proteins. The same electrochemical behaviour was recorded for the type 2 Cu-depleted derivatives, which contain reduced type 3 Cu, whereas the apoproteins were electrochemically inactive. Under aerobic conditions the catalytic current intensity of holoprotein voltammograms increased up to approx. 2-fold at a low scanning rate, with unchanged redox potentials. The voltammograms of type 2 Cu-depleted proteins and of apoproteins were unaffected by the presence of oxygen. This suggests that electron uptake at the electrode surface involves type 1 Cu and that only in the presence of oxygen is the intramolecular electron transfer to other protein sites rapid enough to be observed. The analogy with available kinetic results is discussed.
Assuntos
Ascorbato Oxidase/química , Oxirredutases/química , Eletroquímica , Eletrodos , Enzimas Imobilizadas , Ouro , Concentração de Íons de Hidrogênio , Cinética , Lacase , Membranas , Compostos Organofosforados , Oxirredução , TermodinâmicaRESUMO
Two different Cu2+-depleted derivatives of bovine serum amine oxidase (BSAO) have recently been prepared, which contain about 0.5 mol/dimer of phenylhydrazine-reactive topa quinone (TPQ) cofactor and, depending on the reagents used, about 0.2 or 0.7 residual Cu2+/dimer [Agostinelli, De Matteis, Sinibaldi, Mondovi and Morpurgo (1997) Biochem. J. 324, 497-501]. The benzylamine oxidase activity of both derivatives was <5% and increased up to approximately 20% on incorporation of Co2+, irrespective of the residual Cu2+ content, which was unaffected by the treatment according to atomic absorption and ESR spectroscopy. The residual Cu2+ ions appeared to be distributed one per dimer and to be bound to inactive subunits, whereas Co2+ was bound to active subunits. The change in the active site had an appreciable influence on the kinetic behaviour. With several amines, the kinetic parameters, Km and kc, measured for Co2+-BSAO were different from those for native BSAO. This excludes the possibility that the catalytic activity was due to residual Cu2+. Furthermore, Co2+ restored to nearly native level the intensity of the TPQ 480 nm band and the reactions with phenylhydrazine or benzylhydrazine, which had been slowed down or abolished, respectively, in Cu2+-depleted samples. The CD spectrum, measured for the derivative with low Cu2+ content, was compatible with Co2+ binding to the copper site. The amine oxidase activity of the Co2+ derivative, which cannot form a semiquinone radical as an intermediate of the catalytic reaction, strongly suggests that the Cu+-semiquinone is not an obligatory intermediate of BSAO catalytic pathway.
Assuntos
Amina Oxidase (contendo Cobre) , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Animais , Benzilaminas/química , Catálise , Bovinos , Cobalto/química , Cobre/química , Cianetos/química , Ditionita/química , Metaloproteínas/química , Relação Estrutura-AtividadeRESUMO
A novel copper-depleted bovine serum amine oxidase (BSAO), in which about half the molecules contained the organic cofactor in the oxidized form, was prepared by adding a reductant in anaerobic conditions to the cyanide-reacted protein. The CuI-semiquinone formed in these conditions reoxidizes after the removal of copper. The inactive derivative was reduced by benzylamine at approx. 1/1000 the rate of BSAO. The pseudo-first-order reaction was preceded by the formation of a protein-benzylamine complex with dissociation constant, Kd, of 4.9+/-0.5 mM, similar to the Km of BSAO (2.2 mM). Also the reactions with phenylhydrazine and benzohydrazide were considerably slower than in holo-BSAO, whereas the reactions with p-pyridine-2-ylphenylacetohydrazide, containing a longer aromatic tail, and semicarbazide, lacking an aromatic moiety, were less severely affected. Removal of copper had no effect on the optical spectra of BSAO and of most adducts, containing the cofactor in quinol form, showing that copper is bound to neither the oxidized nor the reduced cofactor. Benzylhydrazine did not produce optical effects but was tightly bound, as inferred from its inhibitory effect on reaction with other molecules. Substrate and inhibitors might bind a hydrophobic pocket at some distance from the quinone, probably near the protein surface, with their affinity depending on the hydrophobic character and pKa. The binding, which is not greatly influenced by copper removal, probably induces a copper-dependent change of conformation, 'opening' a pathway to the active site buried in the protein interior.
Assuntos
Amina Oxidase (contendo Cobre) , Cobre/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Animais , Benzilaminas/metabolismo , Catálise , Bovinos , Di-Hidroxifenilalanina/análogos & derivados , Di-Hidroxifenilalanina/química , Inibidores Enzimáticos/farmacologia , Fabaceae/enzimologia , Hidrazinas/farmacologia , Cinética , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/sangue , Fenil-Hidrazinas/farmacologia , Proteínas de Plantas/química , Plantas Medicinais , Piridinas/farmacologia , Semicarbazidas/farmacologia , Especificidade da EspécieRESUMO
The organic cofactor of bovine serum amine oxidase was identified as 2,4,5-trihydroxyphenylalanine quinone by means of the phenylhydrazine adduct [Janes, S. M., Mu, D., Wemmer, D., Smith, A. J., Kaur, S., Maltby, D., Burligame, A.L. & Klinman, J.P. (1990) Science 248, 981-987]. A still debated question is, however, whether the dimeric protein binds two mol phenylhydrazine/mole or only one, that is whether it actually contains two identical independent carbonyl cofactors. This matter is addressed in the present study by means of the protein reactions with phenylhydrazine and other inhibitors such as semicarbazide and p-pyridine-2-yl-phenylacetohydrazide. The two latter reagents were found to bind in two steps, one mole/mole dimer in the first step with loss of catalytic activity but only about (0.10-0.35 mol/mol) in the second one. Similar results were obtained by either optical spectroscopy or by reverse-phase HPLC of the labelled peptides produced on proteolysis. Irrespective of the inhibitor nature and reacted amount, all adducts formed on proteolysis a single labelled peptide, of same 25-amino-acid composition, showing that the same cofactor is present in both subunits, in the same stretch of the polypeptide chain. The slow reaction of the second cofactor may be related to slow conformational equilibria, which are established after the first cofactor has reacted and are probably mediated by a change of the hydrogen bond pattern. The conformers spectroscopic properties suggest that they differ in whether the cofactor does or does not directly interact with copper.
Assuntos
Amina Oxidase (contendo Cobre) , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/sangue , Sequência de Aminoácidos , Animais , Bovinos , Hidrólise , Dados de Sequência Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Desnaturação Proteica , Especificidade por SubstratoRESUMO
The thermal denaturation of laccase from the Japanese lacquer tree (Rhus vernicifera) was studied by differential scanning calorimetry. The endotherms of holo-laccase, type 2-Cu-depleted laccase and apo-laccase were deconvoluted into two independent two-state transitions, providing evidence for a domain structure of the protein. The correlation of the two transitions with the bleaching of copper optical bands and the decrease of the transitions' enthalpy on Cu removal show that the process involves the denaturation of Cu sites. No detectable unfolding of secondary structure was observed, since the thermal transitions, characterized by low overall specific enthalpy, did not modify either the laccase c.d. spectra in the beta-fold region or the maximum wavelength of the fluorescence emission. On chemical denaturation, however, the emission was red-shifted by about 20 nm. The laccase behaviour is substantially different from that of stellacyanin, a protein containing a single blue Cu ion, in which the thermal transition had higher specific enthalpy and induced a large change of the c.d. spectrum in the beta-fold region. The laccase denaturation behaviour is similar to that of ascorbate oxidase from zucchini (courgette; Cucurbita pepo) [Savini, D'Alessio, Giartosio, Morpurgo and Avigliano (1990) Eur. J. Biochem. 190, 491-495], suggesting a structural analogy. In both proteins heating may cause a change of tertiary structure through modifications of Cu co-ordination with loosening of the bonds between the structural domains at the interface of which the trinuclear Cu cluster is located.
Assuntos
Ascorbato Oxidase/química , Oxirredutases/química , Plantas/enzimologia , Estabilidade Enzimática , Temperatura Alta , Lacase , Desnaturação ProteicaRESUMO
Half-copper-depleted and fully copper-depleted amine oxidase from bovine serum were reconstituted with either copper or cobalt. All samples were studied by high-sensitivity scanning calorimetry, by enzyme activity analysis, and by reactivity with phenylhydrazine. The calorimetric profile of the protein was strongly modified by the removal of a single Cu ion approximately to the same extent as by complete copper removal, in agreement with the loss of over 80% enzymic activity. The thermograms of metal-reconstituted species showed a marked similarity with that of the native enzyme, irrespective of whether copper or cobalt was present. Reactivity with phenylhydrazine and enzymic activity measurements showed that in cobalt-substituted amine oxidase the organic cofactor was reactive and the enzyme was catalytically competent, although kinetically less efficient. These observations agree both with previous findings on the protein half-site reactivity and with previous suggestions for a copper conformational role in bovine serum amine oxidase, namely of maintaining a functional conformation at the active site.
Assuntos
Amina Oxidase (contendo Cobre) , Cobalto/química , Cobre/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Animais , Calorimetria , Bovinos , Cobalto/metabolismo , Cobre/metabolismo , Estabilidade Enzimática , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/sangue , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Fenil-Hidrazinas/metabolismo , Conformação Proteica , Desnaturação Proteica , Temperatura , TermodinâmicaRESUMO
The electronic properties of 2,4,5-trihydroxyphenylalanine quinone (TPQ), the cofactor of bovine serum amine oxidase [Janes, S. M., Mu, D., Wemmer, D., Smith, A. J., Kaur, S., Maltby, D., Burlingame, A. L., & Klinman, J. P. (1990) Science 248, 981-987], and some adducts with hydrazines were investigated by means of low-molecular-weight models and semiempirical molecular orbital calculation methods. The enzyme visible band was assigned to the first pi-->pi* transition of the cofactor in p-quinonic form, with the C-4 hydroxyl ionized and hydrogen bonded either to a water molecule or to a basic protein residue. The spectra of the protein adducts with some substituted hydrazines were well accounted for by assuming the inhibitor bound to the C-5 carbonyl, usually in azo form. The adduct with the unsubstituted hydrazine was instead assigned an o-quinone hydrazone form, stabilized by an internal hydrogen bond between the amino group and the ortho carbonyl oxygen, a larger electron delocalization, and formation of a hydrogen bond at the C-6 ionized hydroxyl. On the basis of these assignments, the reaction of the protein with benzylhydrazine [Morpurgo, L., Agostinelli, E., Muccigrosso, J., Martini, F., Mondovi, B., & Avigliano, L. (1989) Biochem. J. 260, 19-25] was rewritten. All examined electronic transitions, though highly sensitive to cofactor ionization and hydrogen bonding, could be accounted for without introducing perturbations due to copper. This confirms that copper is not within bonding distance of the oxidized cofactor.
Assuntos
Amina Oxidase (contendo Cobre) , Coenzimas/química , Di-Hidroxifenilalanina/análogos & derivados , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Animais , Benzoquinonas/química , Bovinos , Cobre/química , Di-Hidroxifenilalanina/química , Elétrons , Hidrazinas/química , Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/sangue , Solventes , Espectrofotometria InfravermelhoRESUMO
Aromatic hydrazides of the general formula NH2NHCO(CH2)nC6H4R were covalently bound by bovine serum amine oxidase (BSAO), giving rise to optical and CD absorptions at 350-400 nm. Benzohydrazides (n = 0) reacted slowly, in the ratio of one per dimeric protein molecule, like semicarbazide. Phenylacetohydrazides (n = 1) and phenylpropionic hydrazides (n = 2) reacted instead in the ratio of two per dimer, one molecule at a much faster rate than the other. The fast reaction correlated with the loss of enzymatic activity. The contribution to the optical absorbance of either molecule was identical, but only the first one produced a CD band, the wavelength and sign of which were determined by the number n of methylene groups in the hydrazide. In n = 1 and n = 2 compounds, the reaction was faster as the R substituent became more hydrophobic (triazolyl less than imidazolyl less than phenyl), suggesting a specific interaction with the protein matrix. Phenylhydrazine was found to react with the native enzyme in the ratio of only one per protein dimer. However, one phenylhydrazine was also slowly bound by most 1:1 enzyme-hydrazide adducts, with the formation of ternary derivatives. Phenylhydrazine formed the usual intense band at 447 nm with n = 1 and n = 2 hydrazide-BSAO adducts and a weaker, blue-shifted band with the adducts of semicarbazide and of some n = 0 hydrazides. In both cases, the hydrazide absorption band was unaffected. Competition was observed with other benzohydrazides and with the second molecule of n = 1 compounds. A half-site mechanism appears to be operative, the second site being always less reactive than the first. Reactivity and adduct conformation were also affected by N,N-diethyldithiocarbamate, a powerful enzyme inhibitor that binds copper.
Assuntos
Amina Oxidase (contendo Cobre) , Hidrazinas/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/sangue , Fenil-Hidrazinas/metabolismo , Semicarbazidas/metabolismo , Animais , Sítios de Ligação , Bovinos , Dicroísmo Circular , Cobre/metabolismo , Ditiocarb/metabolismo , Ditiocarb/farmacologia , Substâncias Macromoleculares , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/antagonistas & inibidores , Conformação Proteica/efeitos dos fármacos , EspectrofotometriaRESUMO
The susceptibility of native, type-2 Cu-depleted and fully Cu-depleted ascorbate oxidase to thermal and chemical denaturation has been probed by differential scanning calorimetry, fluorimetry and circular dichroism. The data indicate that copper affects the stability, but not the protein conformation. The unfolding of ascorbate oxidase is characterized by a single endotherm. Calorimetric domains revealed by deconvolution are consistent with the domains identified by X-ray crystallography.
Assuntos
Ascorbato Oxidase/metabolismo , Cobre/metabolismo , Guanidinas/farmacologia , Oxirredutases/metabolismo , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Estabilidade Enzimática , Guanidina , Temperatura Alta , Plantas/enzimologia , Conformação Proteica , Desnaturação Proteica , Espectrometria de FluorescênciaRESUMO
The role of copper in bovine serum amine oxidase was investigated by studying the effect of copper-binding inhibitors on the reactions of the pyrroloquinoline quinone carbonyl and on the reaction with oxygen. Hydrazines and hydrazides were used as carbonyl reagents and one of the hydrazines, benzylhydrazine, which was found to behave as a pseudo-substrate, was used to probe the reaction with oxygen. The presence of N,N-diethyldithiocarbamate, a chelator that binds copper irreversibly, did not prevent the reactions at the carbonyl, but slowed down their rate and modified the conformation of the adducts. The same happened to the reaction with oxygen, which was slowed down but not abolished. Copper, which was never seen in the reduced state, thus appears to control all reactions without being directly involved in the binding of either hydrazines or oxygen. The enzyme functionality was in fact preserved upon substitution of copper with cobalt. The specific activity of the cobalt-substituted enzyme was only reduced to about 40% the native amine oxidase value. This is the first case so far in which the role of copper can be performed by a different metal ion.
Assuntos
Amina Oxidase (contendo Cobre) , Cobre/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/sangue , Animais , Sítios de Ligação , Bovinos , Dicroísmo Circular , Cobalto/metabolismo , Hidrazinas , Técnicas In Vitro , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Espectrofotometria UltravioletaRESUMO
Bovine serum amine oxidase is inhibited by benzylhydrazine (BHy), but recovers full activity after a few hours incubation [Hucko-Haas & Reed (1970) Biochem. Biophys. Res. Commun. 38, 396-400]. The first phase of the process, requiring about 15 min, was found to consist of a mechanism-based hydrazine-transfer reaction leading to formation of the hydrazine-bound enzyme, benzaldehyde and H2O2. At variance with the enzymic process, the reaction with O2 preceded the benzaldehyde release. Two reaction intermediates could be characterized by optical spectroscopy and were assigned as the azo derivative and the benzaldehyde hydrazone, the latter one probably being involved in the reaction with O2. No reduction of Cu was detected at any stage. The hydrazine adduct could also be obtained by stoichiometric reaction of hydrazine with the native enzyme. The decay of this species occurred in about 8 h and was not studied in detail. The Cu-binding inhibitor NN-diethyldithiocarbamate affected the BHy reaction by stabilizing the benzaldehyde hydrazone form as against the azo derivative and the reaction with O2. However, under these same conditions the initial spectroscopic properties of the diethyldithiocarbamate adduct were recovered if the oxidase was left overnight. The reaction with O2 was abolished only upon removal of at least one Cu atom from the enzyme. On the basis of the failure to detect any change of Cu redox state and the enzyme behaviour in the presence of inhibitors, a reaction mechanism involving the formation of a hydroperoxy intermediate, as in the FAD-containing enzymes, is tentatively proposed.
Assuntos
Amina Oxidase (contendo Cobre) , Hidrazinas/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Animais , Bovinos , Ditiocarb , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/antagonistas & inibidoresRESUMO
The carbonyl cofactor of bovine serum amine oxidase, recently identified as pyrroloquinoline quinone [Ameyama, Hayashi, Matsushita, Shinagawa & Adachi (1984) Agric. Biol. Chem. 48, 561-565; Lobenstein-Verbeek, Jongejan, Frank & Duine (1984) FEBS Lett. 170, 305-309], reacts stoichiometrically and irreversibly with hydrazides of phenylacetic acid and of benzoic acid. With the phenylacetic hydrazides a reversible intermediate step was detected by competition with substrate, carbonylic reagents or phenylhydrazine, a typical inhibitor of the enzyme. All hydrazides form an intense broad band with maximum absorbance in a narrow wavelength range (350-360 nm), irrespective of the acyl group, suggesting that the transition is located on the organic cofactor. A different situation is found with some phenylhydrazines, where extended conjugation can occur between the cofactor and the phenyl pi-electron system via the azo group, as shown by the lower energy and higher intensity of the transition. In this case the transition is sensitive to substituents in the phenyl ring. The c.d. spectrum of the adducts is influenced by the type of hydrazide (derived from phenylacetic acid or benzoic acid), by pH and by NN-diethyldithiocarbamate binding to copper, probably as a result of shifts of equilibria between hydrazone-azo tautomers.