STOPPED-FLOW KINETIC-STUDIES OF THE REACTION OF BARLEY ALPHA-AMYLASE SUBTILISIN INHIBITOR AND THE HIGH PI BARLEY ALPHA-AMYLASE

The interaction of alpha-amylase/subtilisin inhibitor (BASI) from barl ey seeds and the high pI barley alpha-amylase (AMY2) de novo synthesiz ed during seed germination, has been studied at pH 8.0, 25 degrees C, using stopped-flow fluorescence spectroscopy, equilibrium fluorescence titration and kinetic analysis of the displacement of BASI from the B ASI-AMY2 complex by the substrate blue starch, The results are in acco rdance with a two-step reaction model: [GRAPHICS] The resulting values of the kinetic parameters were: k(2)/K-1 = (1.0 +/- 0.2) x 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) x 10(-5)s(-1), and the overall dissociation constant K-d = (0.7 +/- 0.1) x 10(-10) M. BASI thus is best characterized as a fast r eacting, tight-binding inhibitor of AMY2.