Studies were made on the ultraviolet difference-spectra of glucoamylase from Rhizopus niveus [EC 3.2.1.3] specifically produced by the substrate maltose and the inhibitors, glucose, glucono-1: 5-lactone (gluconolactone), methyl beta-D-glucoside, cellubiose, and cyclohexa-, and cyclohepta-amyloses. Of these, maltose and gluconolactone produced characteristic difference spectra with a trough near 300 nm. Based on studies with a model compound for a tryptophan residue, Ac-Trp, this trough was attributed to the effect of a negative charge upon the tryptophan residue. From the concentration dependency of the difference spectra, the dissociation constants of the complexes between the enzyme and maltose, glucose, and gluconolactone were evaluated to be 1.2 mM, 51 mM, and 1.5 mM, respectively. These values are in good agreement with the values of Km or K1 obtained from the steady-state kinetics. The difference-spectrophotometric data suggested that referring to the values of subsite affinities of glucoamylase, maltose, and gluconolactone occupy mainly Subsite 1, where the non-reducing-end glucose residue of a substrate is bound in a productive form and that a tryptophan residue with shows a trough near 300 nm in difference spectra is located in this subsite.

The difference spectra of liquefying alpha-amylase (EC 3.2.1.1) from B. subtilis upon the addition of a slowly reacting substrate, maltotriose, were measured to investigate specific binding of the substrate to the enzyme. The spectra produced by maltotriose were attributed to one tryptophan and one tyrosine residues on the basis of analysis of their shape and magnitude. From the dependence of the difference absorption upon the concentration of maltotriose, the dissociation constant of the maltotriose-enzyme complex was determined to be 170(+/- 20) mM, which is in good agreement with the Michaelis constant, Km obtained from the steady-state kinetics. The difference spectrum characteristic of a tryptophan residue was significantly decreased by the chemical modification of a trytophan residue with N-bromosuccinimide.