Kinetic study on chemical modification of Taka-amylase A. II. Ethoxycarbonylation of histidine residues

Journal of Biochemistry
Y KitaT Watanabe

Abstract

The modification of Taka-amylase A (TAA) [EC 3.2.1.1] of Aspergillus oryzae by diethylpyrocarbonate (DEP) was carried out at 25 degrees 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 alpha-maltoside (phi alpha M). The kinetic parameters of the modified TAA for several substrates and analogs were determined at 25 degrees 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 k0 value. Thus, some of the histidine residues in TAA are suggested to play an important role in the enzyme catalytic function.

Citations

Aug 1, 1988·Journal of Protein Chemistry·E A MacGregor
Jan 1, 1993·Critical Reviews in Oral Biology and Medicine : an Official Publication of the American Association of Oral Biologists·F A ScannapiecoM J Levine

Related Concepts

Mégamylase
Amylases
Aspergillus
Aspergillus oryzae
Diethyl Pyrocarbonate
Formic Acids
Histidine
Plasma Protein Binding Capacity

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