Studies of the Interaction of Substrate Analogues with Bacterial Liquefying α-Amylase* by Means of Spectrophotometry and Steady State Kinetics

(1)The inhibition by β-cyclodextrin (cyclomaltoheptaose), maltose, and glucose of the hydrolysis of amylose by bacterial liquefying α-amylase [EC 3. 2. 1.1] was studied kinetically. The type of the inhibition was competitive for all the inhibitors employed, and the inhibitor constant K1's determined were 0. 018 M for β-cyclodextrin, 0. 072 M for maltose and 0. 38M for glucose. 2) The number of exposed tryptophan residues was estimated by solvent perturbation technique, in which various organic perturbants were used. It was found that 8 to 9 tryptophan residues are exposed although the enzyme has 15 tryptophan residues in total. 3) Difference spectra of the enzyme in the presence of β3-cyclodextrin, maltose, or glucose were measured to investigate their specific binding with the enzyme. In the presence of β-cyclodextrin, a characteristic difference spectrum was observed. The difference spectrum was attributed to one tryptophan and one tyrosine residues on the basis of the analyses of its shape and magnitude. On the basis of the difference-spectral titration at 291–293 mμ, the dissociation constant of β-cyclodextrin-enzyme complex was determined to be 0. 018 M, which was in good agreement with K1 value obtained from kinetic studies. Maltose and glucose, however, did not show any characteristic difference spectrum other than that arising from common solvent perturbation, although they formed complexes with the enzyme as inferred from their competitive type of inhibition. 4) The comparison of K1 values for the three competitive inhibitors with each other indicated that β-cyclodextrin covered an additional area (subsite) at the active site of the enzyme and that the area was not covered either by glucose or by maltose. By combination of the results obtained by means of kinetic and difference-spectral techniques, it was suggested that one tryptophan and one tyrosine residues are located in the subsite of the enzyme, in which the subsite is covered by βJ-cyclodextrin but not by maltose or glucose.