Structure and Function of D-Amino-Acid Oxidase

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Abstract
Chemical modification of arginine residues of D-amino-acid oxidase [EC 1.4.3.3, D-amino-acid: oxygen oxidoreductase (deaminating)] was brought about at pH8.1 and at room temperature using glyoxal as their blocking agent, in the absence or presence of benzoate which forms a 1:1 complex with the enzyme on the molecular basis of 50,000. The free enzyme was inactivated partially in proportion to the extent of modification of its arginine residues, and finally completely when reactive 18 out of 28 residues per molecule were modified. On the other hand, considerable resistance against inactivation was found when the enzyme previously formed a complex with benzoate; about 50% of the enzyme activity still remained even after the modification of reactive 17 residues per molecule. The enzyme inactivated in the absence of benzoate completely lost the ability to form a complex with benzoate, whereas in the presence of benzoate the enzymes whose activities were lowered to about 50% by glyoxal still kept the full ability of complexing with benzoate. The yellow color of the modified enzyme preparation was completely bleached on addition of D-alanine under anaerobic conditions. No significant difference was found between the apparent Km value of the intact enzyme and that of the 50%-active enzyme. The apparent Vmax value of this modified enzyme, however, was lowered to about a half that of the intact enzyme. Optical activity of the flavin chromophore of the modified enzyme was found to be essentially identical with that of the intact enzyme. These results indicate the complete protection of the binding sites of the enzyme by complexing with benzoate and, therefore, strongly suggest the presence of a side group in the protein molecule, most probably one specific arginine residue that contributes to the enzyme action through its binding with the carboxylate group of the substrate.

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