Kinetics and Energetics of the Binding between Barley α-Amylase/Subtilisin Inhibitor and Barley α-Amylase 2 Analyzed by Surface Plasmon Resonance and Isothermal Titration Calorimetry

The kinetics and energetics of the binding between barley α-amylase/subtilisin inhibitor (BASI) or BASI mutants and barley α-amylase 2 (AMY2) were determined using surface plasmon resonance and isothermal titration calorimetry (ITC). Binding kinetics were in accordance with a 1:1 binding model. At pH 5.5, [Ca2+] = 5 mM, and 25 °C, the k on and k off values were 8.3 × 10+4 M-1 s-1 and 26.0 × 10-4 s-1, respectively, corresponding to a K D of 31 nM. K D was dependent on pH, and while k off decreased 16-fold upon increasing pH from 5.5 to 8.0, k on was barely affected. The crystal structure of AMY2−BASI shows a fully hydrated Ca2+ at the protein interface, and at pH 6.5 increase of [Ca2+] in the 2 μM to 5 mM range raised the affinity 30-fold mainly due to reduced k off. The K D was weakly temperature-dependent in the interval from 5 to 35 °C as k on and k off were only increasing 4- and 12-fold, respectively. A small salt dependence of k on and k off suggested a minor role for global electrostatic forces in the binding and dissociation steps. Substitution of a positively charged side chain in the mutant K140L within the AMY2 inhibitory site of BASI accordingly did not change k on, whereas k off increased 13-fold. ITC showed that the formation of the AMY2−BASI complex is characterized by a large exothermic heat (ΔH = −69 ± 7 kJ mol-1), a K D of 25 nM (27 °C, pH 5.5), and an unfavorable change in entropy (−TΔS = 26 ± 7 kJ mol-1). Calculations based on the thermodynamic data indicated minimal structural changes during complex formation.