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

The 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: k 2/ K 1 = (1.0 ± 0.2) × 10 6 M −1·s −1, K 1 = 0.4 ± 0.21 mM, k 2 = 320 ± 150 s −1, k −2 = (7.2 ± 0.6) × 10 −5s −1, and the overall dissociation constant K d = (0.7 ± 0.1) × 10 −10 M. BASI thus is best characterized as a fast reacting, tight-binding inhibitor of AMY2.