Mechanism of enzyme action II. Characterization of the purple intermediate in the anaerobic reaction of d-amino-acid oxidase with d-alanine

Abstract

1. 1.|The purple intermediate in the anaerobic reduction of d-amino-acid oxidase ( d-amino-acid:O 2 oxidoreductase (deaminating), EC 1.4.3.3) with d-alanine had a broad absorption band in the vicinity of 550 mμ, an extrinsic negative Cotton effect in optical rotatory dispersion having a trough at 430 mμ, an inflexion point close to 400 mμ and a peak at 380 mμ, but no significant electron spin resonance signal. 2. 2.|After the preparation had been aged in the dark at 5°, a new absorption peak appeared at 492 mμ, accompanying the gradual disappearance of the broad absorption band peaking at 550 mμ and the appearance of an electron spin resonance signal. The increase in A 492 m μ A 550 mμ was proportional to the decrease in the amplitude of the trough at 430 mμ, and the increase in the concentration of unpaired electrons. 3. 3.|The purple intermediate changed into the benzoate complex of the oxidized form when the preparation was treated with an excess of triturated sodium benzoate under anaerobic conditions, indicating that this intermediate is a molecular complex between the enzyme and substrate that can be replaced by benzoate. However, in the aged preparation only part of the total enzyme changed into the benzoate complex, and no significant change was found in the concentration of unpaired electrons before and after the addition of benzoate; this indicates the presence of two species in the aged material, the molecular complex and the semiquinoid enzyme which is derived from the former. 4. 4.|The conversion from the diamagnetic purple intermediate into the paramagnetic species occurred without light, although light accelerated this conversion. This phenomenon is probably explained in terms of a charge separation process of shared electrons between the enzyme and the substrate moiety.