Studies on the Elimination Reaction of d-Amino Acid Oxidase with α-Amino-β-chlorobutyrate
Year: 1973
Type: article
Abstract: d-Amino acid oxidase catalyzes the nonoxidative conversion of α-amino-β-chlorobutyrate to α-ketobutyrate, but does not catalyze its oxidation to β-chloroketobutyrate. The elimination reaction requires the oxidized flavoprotein, i.e. it does not occur with the apoenzyme or when the enzyme-bound flavin is reduced. The occurrence of this elimination reaction supports the proposal that removal of the substrate α proton is an early step in reactions catalyzed by d-amino acid oxidase. Addition of α-amino-β-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: ki, 2000 min-1; kii, 40; and kiii, ∼1. (ki is dependent on substrate concentration. The value given is the limiting value at infinite substrate concentration.) When an α deuterated substrate is used, ki shows a deuterium isotope effect of about 5. Therefore, formation of Complex I must involve breaking of the substrate α 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-1. From experiments carried out with α-amino-β-chloro[α-2H]butyrate and in 2H2O, it is concluded that the action of d-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. (b) The β position of the enamine is protonated on the enzyme to form α-iminobutyric acid, which is then released and hydrolyzes to α-ketobutyrate. The proton which is added to the β position is originally derived from the α position of the substrate. The enzyme thus has the capability of sequestering the α proton of the substrate so that it does not exchange with the solvent. The relative extent to which these two processes (enamine reprotonation versus release) occur is temperature-dependent.
Source: Journal of Biological Chemistry
Institutions: University of Michigan, Brandeis University
Language: English
Subfield: Biochemistry
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W1511801414