Amine Oxidase from Lentil Seedlings: Energetic Domains and Effect of Temperature on Activity

Abstract

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).

Copper/topaquinone-containing amine oxidase from lentil seedlings and bovine plasma: Catalytic mechanism and energetic domains

“…The thermal denaturation that starts at 55 °C corresponds to decreasing activity. The DSC curve for LSAO shows a small and broad transition below 55 °C, the enthalpy of which is consistent with a conformational change in the native enzyme, whereas for BSAO there is negligible variation in the same range of temperature [37,39], which is in agreement with the specific activity variation studies previously reported [40]. The deconvolution of the excess molar heat capacity reveals in LSAO and BSAO three subpeaks (transitions I-III) and five subpeaks respectively [37,36].…”

“…Thus the Arrhenius plot shows a discontinuity which depicts two distinct linear lines with different steepness [37]. The change in LSAO activity at different temperatures and the Arrhenius plot show a similar behaviour as the curves measured for BSAO [38].…”

“…The DSC curve for LSAO shows a small and broad transition below 55 °C, the enthalpy of which is consistent with a conformational change in the native enzyme, whereas for BSAO there is negligible variation in the same range of temperature [37,39], which is in agreement with the specific activity variation studies previously reported [40]. The deconvolution of the excess molar heat capacity reveals in LSAO and BSAO three subpeaks (transitions I-III) and five subpeaks respectively [37,36]. In LSAO the subpeaks (energetic domains, transitions I,II and III) [41] may belong to the three hypothesized structural domains for each subunit of LSAO (D3 , D2 and D4 respectively) [5].…”

“…The deconvolution of the excess molar heat capacity reveals in LSAO and BSAO three subpeaks (transitions I-III) and five subpeaks respectively [37,36]. In LSAO the subpeaks (energetic domains, transitions I,II and III) [41] may belong to the three hypothesized structural domains for each subunit of LSAO (D3 , D2 and D4 respectively) [5]. There are great differences in the energetic domains in the DSC profiles for LSAO and BSAO.…”

“…The temperature dependence of the molar heat capacity (C p ) of native LSAO, obtained by calorimetric results, shows that a broad transition starts at 16 °C and ends at 37 °C, whereas the thermal profile begins to increase at 45 °C, with a T m = 65.18 °C [37]. The thermal denaturation that starts at 55 °C corresponds to decreasing activity.…”