On the basis of the “subsite” concept, we tried to elucidate the active-site structure of the glucoamylase from Rhizopus niveus: The subsite structure, that is, the arrangement of the subsites and the affinity of each subsite, of the glucoamylase active-site was evaluated using the steady-state kinetic method described by Hiromi et al. The binding subsites for glucose, gluconolactone, maltose, isomaltose and some maltooligosaccharides were investigated by means of UV-difference spectrophotometry, fluorescence spectrophotometry, and as to steady-state inhibition-kinetics and stopped-flow kinetics. Based on several lines of evidence, it was concluded that the nonreducing-end-glucose-residue of a substrate (in a productive mode) and gluconolactone, a transition-state analogue are bound at Subsite 1, at which one tryptophan residue is located. The tryptophan residue was clearly discriminated with the stopped-flow chemical modification with N-bromosuccinimide.
