Studies on the mechanism of action of D-amino acid oxidase. Evidence for removal of substrate -hydrogen as a proton.

Abstract d-Amino acid oxidase catalyzes the oxidation of β-chloroalanine to chloropyruvate as well as its conversion to pyruvate, a nonoxidative reaction. The product composition depends on oxygen: only pyruvate is produced in 100% N2, chloropyruvate is formed almost exclusively in 100% O2, and mixtures result at intermediate levels. The rate of total keto acid production (pyruvate + chloropyruvate) is independent of oxygen, indicating the rate-determining formation of a common precursor of the two keto acids. Substitution of the α-hydrogen of β-chloroalanine by deuterium and tritium results in kinetic isotope effects which slow down chloroalanine conversion but do not differentially affect product distribution. A similar relationship holds for the deuterium solvent isotope effect with incubations conducted in 2H2O. The substrate kinetic isotope effects indicate that the dissociation of the α-C—H bond of the substrate is rate-determining or at least partially rate-determining in the formation of the common intermediate. Preliminary experiments indicate that l-amino acid oxidase also produces both pyruvate and chloropyruvate from l-chloroalanine. These results suggest that the conversion of chloroalanine to pyruvate and chloropyruvate by d-amino acid oxidase involves a common intermediate in which the α-hydrogen of the substrate has been removed as a proton. It is very probable that the oxidation of other amino acid substrates involves similar intermediates. The intermediate could be a carbanion or an adduct derived from a substrate carbanion and the flavin coenzyme. d-Amino acid oxidase does not catalyze the exchange of the α-hydrogen of chloroalanine and serine with solvent protons under anaerobic conditions. When [α-3H]proline, pyruvate, and NH4+ were incubated anaerobically with the enzyme, no 3H was observed in the alanine formed in such incubations. This indicates that the proton released from the substrate is rapidly released from E·FADII2.