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 (alpha-amylase/subtilisin inhibitor (BASI) or BASI mutants and barley (alpha-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 degreesC, the k(on) and k(off) values were 8.3 x 10(+4) M-1 s(-1) and 26.0 x 10(-4) s(-1), respectively, corresponding to a K-D of 31 nM. KD was dependent on pH, and while koff 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 muM to 5 mM range raised the affinity 30-fold mainly due to reduced koff. The KD was weakly temperature-dependent in the interval from 5 to 35 degreesC 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 (DeltaH = -69 +/- 7 kJ mol(-1)), a K-D of 25 nM (27 degreesC, pH 5.5), and an unfavorable change in entropy (-TDeltaS = 26 +/- 7 kJ mol(-1)). Calculations based on the thermodynamic data indicated minimal structural changes during complex formation.