Reactivity of D-amino acid oxidase with artificial electron acceptors

1. 1.|The fully reduced form of D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3] reacted with a number of electron acceptors and the order of reactivity was: benzoquinone > phenazine methosulfate (PMS) > oxygen > 2,6-dichlorophenolindophenol (DCIP) > methylene blue > ferricyanide. The stoichiometric reaction between the fully reduced enzyme and PMS was established. 2. 2.|A mechanism for the catalytic oxidation of D-arginine by the enzyme using PMS as electron acceptor involved the fully reduced enzyme which was reoxidized by PMS. 3. 3.|When D-alanine was used as substrate, PMS, ferricyanide, DCIP and methylene blue served as electron acceptor. Analyzing the kinetic data of the catalytic oxidation of D-alanine as well as D-α-aminobutyric acid when PMS was used as acceptor, it was revealed that the purple intermediate formed through the reaction of the enzyme with the substrate was involved in the mechanism of the oxidation. The rate of reaction of PMS with the intermediate was fairly small compared with that of PMS with the fully reduced enzyme. 4. 4.|PMS reacted stoichiometrically with the semiquinoid form of the enzyme at a faster rate than with the fully reduced enzyme