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 barley seeds and the high pI barley alpha-amylase (AMY2) de novo synthesized 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 BASI-AMY2 complex by the substrate blue starch. The results are in accordance with a two-step reaction model: [formula: see text] The resulting values of the kinetic parameters were: k2/K1 = (1.0 +/- 0.2) x 10(6) M-1.s-1, K1 = 0.4 +/- 0.21 mM, k2 = 320 +/- 150 s-1, k-2 = (7.2 +/- 0.6) x 10(-5)s-1, and the overall dissociation constant Kd = (0.7 +/- 0.1) x 10(-10) M. BASI thus is best characterized as a fast reacting, tight-binding inhibitor of AMY2.
Sidenius
U
U
Department of Chemistry, Carlsberg Laboratory, Copenhagen, Denmark.
Olsen
K
K
Svensson
B
B
Christensen
U
U
D001467:Hordeum / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D012639:Seeds / Q000378:metabolism; D013997:Time Factors; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000494:pharmacology*; D000516:alpha-Amylases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis
C092772:barley alpha-amylase-subtilisin inhibitor; D014360:Trypsin Inhibitor, Kunitz Soybean; D000516:alpha-Amylases