Study on the interaction between soybean beta-amylase and substrate by the stopped-flow method.

Abstract

The hydrolysis of substrates (maltoheptaose, maltopentaose, and maltotetraose) catalyzed by soybean beta-amylase [EC 3.2.1.2] at pH 5.4 and 25 degrees C was followed by monitoring small changes in the quenching of fluorescence due to tryptophan residues by the stopped-flow method. By analysis of whole time course, the dissociation constants, KdS, of enzyme-substrate and enzyme-product complexes were reasonably evaluated; and the difference in fluorescence intensities per mol between the enzyme-complex (ES or EP) and the free enzyme, delta F, was determined. The molecular activity, k0, was also determined by a new method of half time analysis. The KdS and k0 values are in good agreement with our kinetic data reported previously. The delta Fs of substrates were of smaller magnitude than those of products (G2 and G3), which means that the higher the binding affinity of the ligand is, the smaller the delta F value is. This indicates that at least two tryptophan residues must be located in the active site if the enzyme is rigid, or that if there is only one, the active site must undergo a structural change caused by the binding of ligand.