Difference Spectroscopic Study of the Interaction between Taka-Amylase A and Substrates

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Abstract
1. The difference spectra of Taka-amylase A [EC 3.2.1.1] of Aspergillus oryzae produced by three substrates (α-cyclodextrin, maltotriose, and maltose) showed a clear peak at 293 nm due to the red shift of the tryptophan residues. The dependency of the molar extinction coefficient, Δε, of the difference spectrum at 293 nm on the substrate concentration was investigated at 25°C and pH5.3 (0.08 m acetate buffer). From the titration of Δε with substrate, the dissociation constant, K, of the enzyme-substrate complex and the value of the maximum change in the molar extinction coefficient for saturation of the enzyme with substrate, ΔεBmax, were determined as follows: values of K were 3.5 mM, 35 mM, and 55 min, and those of ΔεBmax were 1, 970, 1, 410, and 730 for α-cyclodextrin, maltotriose, and maltose, respectively. For each substrate, the value of K was in good agreement with that of the Michaelis constant, Km, or inhibitor constant, K1. 2. The difference spectrum produced by ethylene glycol, α nonspecific substance with respect to this enzyme, had a maximum peak at 287 nm, and lacked a clear peak at 293 nm. When this enzyme was almost saturated with α-cyclodextrin (about 77% saturation), the difference spectrum with ethylene glycol decreased in the range of wavelength from 280 nm to 310 nm. The difference between the two difference spectra with ethylene glycol was obtained as a spectrum. The spectrum showed a clear peak at 293 nm and the molar extinction coefficient of this peak was about 1, 000 per 20% ethylene glycol. 3. From the results, we estimated that about three tryptophan residues accessible to solvent exist in the substrate binding site of this enzyme, and that these residues become inaccessible to solvent when the enzyme-substrate (α-cyclodextrin) complex is formed.