Mechanistic Studies on the Reverse Reaction Process of D-Amino Acid Oxidase

Abstract

The reverse reaction of D-amino acid oxidase with chloropyruvate and ammonia was studied. An intermediate in the reverse reaction was observed spectrophotometrically on mixing D-amino acid oxidase with chloropyruvate and ammonia aerobically. This intermediate was visibly green and was stable for more than an hour at room temperature. It had an absorption spectrum characterized by peaks at 382 and 450 nm and a broad band which extended beyond 600 nm. These features were identical with those observed in the aerobic forward reaction of β-chloro-D-alanine with the enzyme. Reduction of the intermediate with NaB³H₄ produced tritiated β-chloroalanine. When a limited amount of D-alanine was added to the intermediate, it was partially reduced, forming another state with spectral features similar to those obtained by partial reoxidation of the D-alanine-reduced enzyme with chloropyruvate and ammonia. When the enzyme in the intermediate obtained by mixing the enzyme with chloropyruvate and ammonia was reduced by treatment with sodium dithionite or by photoreduction in the presence of EDTA and 3-methyllumiflavin, β-chloroalanine was produced, but there was no detectable production of either alanine or pyruvate, which would have been obtained if elimination of chloride had occurred. These results suggest that the green intermediate is a complex of the oxidized enzyme with ketimino acid and that this is a common intermediate in the reverse reaction with chloropyruvate and ammonia and in the forward reaction with β-chloro-D-alanine. To explain the results in the reverse reaction consistently with the forward reaction with β-chloro-D-alanine, a reaction mechanism is proposed in which oxidation or elimination occurs concertedly with abstraction of the α-proton.