Studies on the elimination reaction of D-amino acid oxidase with -amino- -chlorobutyrate. Further evidence for abstraction of substrate -hydrogen as a proton.

D-Amino acid oxidase catalyzes the nonoxidative conversion of oc-amino-P-chlorobutyrate to or-ketobutyrate, but does not catalyze its oxidation to P-chloroketobutyrate. The elimination reaction requires the oxidized flavoprotein, i.e. it does not occur with the apoenzyme or when the enzymebound flavin is reduced. The occurrence of this elimination reaction supports the proposal that removal of the substrate 01 proton is an early step in reactions catalyzed by D-amino acid oxidase. Addition of or-amino-P-chlorobutyrate to D-amino acid oxidase leads to the sequential formation of three spectroscopically recognizable complexes (I, II, and III). All of these complexes have the spectra of oxidized flavin and in addition have long wave length absorbance extending to 800 nm. They are formed with the following rate constants: kl, 2000 min-I; kl~, 40; and &, -1. (kl is dependent on substrate concentration. The value given is the limiting value at infinite substrate concentration.) When an OL deuterated substrate is used, kI shows a deuterium isotope effect of about 5. Therefore, formation of Complex I must involve breaking of the substrate a C-H bond. Complex I could be a complex between flavin and a substrate-derived carbanion, or, if the elimination of HCl is complete at this stage, a complex between substrate-derived enamine and flavin. Formation of Complex I cannot be rate-determining, since the turnover number for the catalytic process is 6 to 9.5 min-I. From experiments carried out with cr-amino-pchloro[oc-2H]butyrate and in 2HZ0, it is concluded that the action of n-amino acid oxidase on the substrate results in the formation of an enzyme-bound enamine which then undergoes two competing reactions: (a) It is released to the medium and spontaneously ketonizes to form ketobutyrate.