Abstract
Interaction between a transition-state analogue, gluconolactone (L), and Aspergillus niger β-glucosidase was studied using the stopped-flow method by monitoring the decrease in the enzyme Trp fluorescence intensity, in relation to the subsite structure of the enzyme. The dependence of kapp, which was evaluated from the reaction curve for the binding of L to the enzyme, on [L]o showed a saturation curve. This result is most reasonably accounted by the two-step mechanism; a fast bimolecular binding process followed by a slow unimolecular process. In the first step, L is transiently bound to subsite 2 and in the second step, it relocates to subsite 1, accompanied by a decrease in fluorescense intensity. Furthermore, the dependence of kapp on [L]o in the presence of galactose was investigated with the same procedure. The result supports the mechanism on the binding of gluconolactone.
