Identity of the Rapidly Appearing Purple Intermediate of D-Amino Acid Oxidase

Abstract

1. The time sequence of the appearance of the purple intermediate in the anaerobic reaction of D-amino acid oxidase [D-amino acid: O₂ oxidoreductase (deaminating), EC 1. 4. 3. 3] with the substrate, revealed the existence of two forms in the oxidized enzyme. The major portion of the enzyme molecules rapidly formed the purple intermediate and the minor portion of the molecules reacted slowly. The aerobic binding of the enzyme with o-aminobenzoate, an inhibitor, gave the same conclusion. 2. Ultracentrifugal analysis indicated that the rapidly reacting and slowly reacting forms corresponded to the dimer and monomer of the oxidized enzyme, respectively. This view was further supported by the fact that the slowly reacting form increased upon lowering the enzyme concentration. 3. The spectrum obtained during the rapid phase of the anaerobic reaction of the enzyme with the substrate was a sum of the spectrum of the purple intermediate produced by the dimer and the spectrum of the unreacted oxidized enzyme of monomeric form. These results led us to the conclusion that the purple intermediate produced by the dimer is identical with the purple complex crystallized earlier in this laboratory.