RESUMO
The thermal stability of copper/quinone containing amine oxidases from Euphorbia characias latex (ELAO) and lentil seedlings (LSAO) was measured in 100 mM potassium phosphate buffer (pH 7.0) following changes in absorbance at 292 nm. ELAO was shown to be about 10 degrees C more stable than LSAO. The dissociative thermal inactivation of ELAO was studied using putrescine as substrate at different temperatures in the range 47-70 degrees C, and a "conformational lock" was developed using the theory pertaining to oligomeric enzyme. Moreover ELAO was shown to be more stable towards denaturants than LSAO, as confirmed by dodecyl trimethylammonium bromide denaturation curves. A comparison of the numbers of contact sites in inter-subunits of ELAO relative to LSAO led us to conclude that the higher stability of ELAO to temperature and towards denaturants was due to the presence of larger number of contact sites in the conformational lock of the enzyme. This study also gives a putative common mechanism for thermal inactivation of amine oxidases and explains the importance of C-terminal conserved amino acids residues in this class of enzymes.
Assuntos
Amina Oxidase (contendo Cobre)/química , Euphorbia/química , Temperatura Alta , Látex/metabolismo , Lens (Planta)/enzimologia , Conformação Proteica , Plântula/enzimologia , Amina Oxidase (contendo Cobre)/genética , Amina Oxidase (contendo Cobre)/metabolismo , Sequência de Aminoácidos , Animais , Estabilidade Enzimática , Lens (Planta)/anatomia & histologia , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Desnaturação Proteica , Putrescina/metabolismo , Alinhamento de SequênciaRESUMO
Copper/TPQ amine oxidases from mammalian and plant sources have shown many differences in substrate specificity and molecular properties. In this work the activity of lentil seedling amine oxidase was followed at various temperatures in 100 mM potassium phosphate buffer, pH 7, using benzylamine as substrate. The discontinuous Arrhenius plot of lentil amine oxidase showed two distinct phases with a jump between them. Thermal denaturation of the enzyme, using differential scanning calorimetry under the same experimental conditions, showed a transition at the same temperature ranges in the absence of substrate, indicating the occurrence of conformational changes, with an enthalpy change of about 175.9 kJ/mole. The temperature-induced changes of the activity of lentil amine oxidase are compared with those of bovine serum amine oxidase (taken from the literature).
