Kinetic Study on Chemical Modification of Taka-Amylase A. II. Ethoxycarbonylation of Histidine Residues

Publisher Website
Google Scholar
Abstract
The modification of Taka-amylase A (TAA) [EC 3.2.1.1] of Aspzrgillus oryzae by diethylpyrocarbonate (DEP) was carried out at 25°C and at pH 5.8 (0.1 M acetate buffer). Two out of the six histidine residues were modified with 4.6 mM DEP, and two or three histidine residues were modified with 23 mM DEP. In both cases, one of them was protected from modification by the presence of 15% maltose. The results suggest that two or three out of the six histidine residues are exposed on the surface of the TAA molecule, and one of them exists near the maltose binding site. Ethoxycarbonylation of histidine residues of TAA caused loss of the amylase activity and activation of the hydrolysis of phenyl a-maltoside (øaM). The kinetic parameters of the modified TAA for several substrates and analogs were determined at 25°C and at pH 5.3 (0.08 M acetate buffer). From the results, it was found that this alteration of the enzyme activity by the modification was not due to a change in km value but to a change in ko value. Thus, some of the histidine residues in TAA are suggested to play an important role in the enzyme catalytic function.