Studies on the subsite structure of amylases. II. Difference-spectrophotometric studies on the interaction of maltotriose with liquefying alpha-amylase from Bacillus subtilis.
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.
D000681:Amylases / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites; D007700:Kinetics; D008433:Mathematics; D009844:Oligosaccharides; D011485:Protein Binding; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet
D009844:Oligosaccharides; D000681:Amylases