2.5 Enzyme assays of SuSys
The SuSy activity in the sucrose cleavage direction was measured with the standard reaction mixture containing 50 mM HEPES-NaOH pH 7.0, 2 mM UDP, 200 mM sucrose, and appropriate amount of purified enzyme in a final volume of 50 μL. Reactions were carried out at 37 °C for 5 min and stopped by heating at 95 °C for 2 min, and control experiments were performed immediately to check the decomposition of sucrose by the heat treatment. The product fructose was determined by the reduction of NAD+ at 340 nm following the addition of a 150-μL solution that contained 50 mM HEPPS-NaOH (pH 7.0), 1 mM MgCl2, 1 mM NAD+, 1 mM ATP, 1 μg hexokinase, 1 μg P-glucose isomerase, and 1 μg glucose-6-P dehydrogenase.[10]One unit of SuSy activity was defined as the amount of enzyme that releases 1 μmol of reducing sugars per minute under the specified conditions.
The pH optimum of SuSy activity in the cleavage direction was determined in the pH ranging from 5.0 to 8.5 at 0.5 pH unit intervals. Buffers used were 50 mM MES-HCl (pH 5.0–7.0) and HEPES-NaOH (pH 7.0–8.5). The initial concentrations of sucrose and UDP in the reaction mixture were 200 mM and 2 mM, respectively.
The temperature profiles were obtained by determining the SuSy activity in the direction of sucrose cleavage from 20 °C to 70 °C. In the evaluation of thermal stability, the enzyme was pre-incubated in 50 mM HEPES buffer (pH 7.0) for 15 min from 22 °C to 60 °C without any substrates, alternatively, with the addition of 200 mM sucrose. After the incubation, the residual activity in the sucrose cleavage direction was checked with the standard assay described above.
The influence of divalent metal ions on SuSy was investigated by measuring the activity in 50 mM HEPES (pH 7.0), 200 mM sucrose, and 2 mM UDP at 37 °C in the presence of 2 mM of MgCl2, CaCl2, NiCl2, CuCl2 or ZnCl2.
The kinetic parameters for sucrose varying from 50 mM to 600 mM at a constant concentration of 2 mM UDP and for UDP varied from 0.05 mM to 5 mM at a constant concentration of 200 mM sucrose were measured at 37 °C in 50 mM HEPES buffer (pH 7.0). K m andV max values were determined by nonlinear regression analysis using the enzyme kinetics component of Origin 2021 software.
All reactions were conducted in triplicate. The relative activity (%) was calculated in terms of that of the maximum activity (100%). Coupled enzymes used for SuSy activity assays were purchased from Shanghai yuanye Bio-Technology Co., Ltd, and all the other reagents were analytical grade and commercially available.