Stopped-flow kinetic studies of the reaction of barley α-amylase/subtilisin inhibitor and the high pI barley α-amylase

AbstractThe interaction of α-amylase/subtilisin inhibitor (BASI) from barley seeds and the high pI barley α-amylase (AMY2) de novo synthesized during seed germination, has been studied at pH 8.0, 25°C, using stopped-flow fluorescence spectroscopy, equilibrium fluorescence titration and kinetic analysis of the displacement of BASI from the BASI-AMY2 complex by the substrate blue starch. The results are in accordance with a two-step reaction model: The resulting values of the kinetic parameters were: k2/K1 = (1.0 ± 0.2) × 106 M−1·s−1, K1 = 0.4 ± 0.21 mM, k2 = 320 ± 150 s−1, k−2 = (7.2 ± 0.6) × 10−5s−1, and the overall dissociation constant Kd = (0.7 ± 0.1) × 10−10 M. BASI thus is best characterized as a fast reacting, tight-binding inhibitor of AMY2