Association and Dissociation of Bacterial α-Amylase Molecule

Abstract

The interaction of Bacillus subtilis α-amylase with zinc ion was studied in 0.1 M sodium chloride and 0.005M calcium acetate by sedimentation experiments. Up to the concentration of zinc ion 1.0×10⁻⁴ schlieren diagrams separate into two peaks and the ratio .of slower to faster component is smaller at an early stage of the centrifuge, but this ratio is larger as the run of the centrifugation increases. The values 4.4–5.2S of the slower component correspond to a monomer form of bacterial α-amylase molecule and values of 6.0–6.2S to a dimer form. At the concentrations of zinc ion more than 2.0×10⁻⁴M, higher degrees of association than dimerization occurred. The effect of pH on the sedimentation velocity was also studied with bacterial α-amylase in 0.1 M sodium acetate, 0.005M calcium acetate and acetic acid. s₂₀ decreases from 6.2S to 4.4S as the pH of the enzyme solution is varied from 6 to 5. It may be confirmed that dimerization or polymerization may occur at imidazol groups of histidyl residues in bacterial α-amylase molecule through chelation by a metal ion. Comparative studies on the native bacterial α-amylase and of the photooxidized bacterial α-amylase in the presence of methylene blue were made by the measurements of sedimentation, ultraviolet absorption, enzymatic activity and optical rotation as well as amino acid analysis. From the measurements of ultraviolet absorption spectrum and of enzymatic activity, the bacterial α-amylase suffers some change in its primary structure, but from the measurement of optical rotatory dispersion the secondary structure of this enzyme is slightly affected by photooxidation. Among twelve histidyl residues in the enzyme molecule, only one or two may be located on the surface and contribute to the association.