A modification of d-amino acid oxidase apoenzyme by pyridoxal 5′-phosphate

Abstract

A modification of d-amino acid oxidase apoenzyme by pyridoxal 5′-phospate and the mechanism of inhibition of the enzyme by this compound are described. Reduction of d-amino acid oxidase apoenzyme with NaBH 4 in the presence of pyridoxal 5′-phosphate resulted in the formation of a reduced Schiff's base between ε-amino amino group of a lysine residue and pyridoxal 5′-phosphate, and 2 moles of pyridoxal 5′-phosphate were fixed per 50 000 g of protein. The modified enzyme had about a half enzymic activity of the native enzyme. The affinity of FAD to the modified apoenzyme was about 10-fold less than that of the native apoenzyme, while the Michaelis constant for d-alanine was only 1.5 times larger than that of the native enzyme. The enzyme was inhibited by 5′-phosphate without reduction by NaBH 4. From the kinetic studies on the inhibition, it was also indicated that the interaction of pyridoxal 5′-phosphate with lysine residues of the apoenzyme caused about 10-fold decrease in the affinity of FAD to the apoenzyme and 43% loss of the enzymic activity.