Kinetics studies on the interactin of rhizopus glucoamylase with maltodextrin and maltotriose,utilizing the absorbance change near 300 nm

MaltodExtrin (high-d.p. malto-oligosaccharides) was found to produce a trough at 303 nm in the difference spectrum of glucoamylase (E.C. 3.2.1.3) from Rhizopus niveus upon binding with the enzyme; this trough disappears upon hydrolysis. The trough, which was ascribed to a change, in the electrostatic environment of a tryptophan residue at the terminal subsite of the enzyme, was found closely related to the formation of the enzyme-substrate complex. The kinetics of binding of maltodextrin and maltotriose to the enzyme were studied at pH 4.5. and 5°, by monitoring the trough by the stopped-flow method. The result was consistent with a two-step mechanism, in which a fast, bimolecular association is followed by a slower, uni-molecular isomerization-process. The latter process involves an environmental change of the tryptophan residue, and is considered to be closely connected to the formation of the productive complex essential for the catalysis.