Abstract
Binding of isomaltose with glucoamylase from Rhizopus niveus was studied statically in the comparison with maltose, by monitoring the intensity change of the enzyme fluorescence. The dissociation constants and the maximum fluorescence intensity decreases of the binary com plexes with the enzyme were determined for isomaltose and maltose, in the presence and absence of gluconolactone, a transition state analogue. The dissociation constant of the enzymeisomaltose complex was found to be larger by a factor of ten than that of the enzyme-maltose complex . It was confirmed that isomaltose and gluconolactone bind to the enzyme independently and form the enzymeisomaltose-gluconolactone ternary complex, whereas gluconolactone and maltose bind to the enzyme competitively. Considering that gluconolactone binds to Subsite 1 of the enzyme active site and that substrates must occupy Subsite 1 in their productive binding modes, these findings suggest that isomaltose binds to the enzyme almost nonproductively, resulting its much slower hydrolytic rate compared with maltose .
