MECHANISM OF ENZYME ACTION .4. ISOLATION BY CRYSTALLIZATION OF FULLY REDUCED FORM OF D-AMINO ACID OXIDASE

1. 1. The fully reduced form of d-amino acid oxidase (d-amino acid:O2 oxido-reductase (deaminating), EC 1.4.3.3), which was produced by anaerobic reduction of the enzyme with excess d-alanine, has been isolated by crystallization. The color of the crystals was pale yellow and their shape was a hexagonal prism or hexagonal prism with bipyramids. 2. 2. The absorption spectra of the crystals and their mother liquor were essentially identical with the spectrum of the enzyme fully reduced with sodium dithionite. The crystals were practically diamagnetic. The mother liquor showed a slight anomaly in optical rotatory dispersion, but it was far less than that found in the purple intermediate complex of the enzyme with the substrate. 3. 3. The crystals were found to be composed of equimolar amounts of the fully reduced enzyme and the intact substrate, d-alanine, indicating the exchange of the product for the intact substrate; a direct evidence for the turnover of substrate on the surface of the enzyme molecule. 4. 4. By mixing the fully reduced enzyme with excess pyruvate and ammonia, the purple intermediate complex was formed. On eliminating the pyruvate in the purple intermediate solution by treatment with hydrogen peroxide, the purple color faded to pale yellow. This indicates the reversible conversion between the purple complex and the fully reduced form. © 1968.