Metabolism of Aromatic Compounds by Microbes

Part VIII. Further Studies of Centisic Acid Oxidase

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1. A highly purified gentisic acid oxidase preparation free from protocatechuic acid oxidase was obtained by the repeated ammonium sulfate precipitations. The specific activity increased up to twenty-fold. This enzyme preparation is free from oxidases of other aromatic compounds, as maleylpyruvate isomerase and fumarylpyruvate hydrolyase.
2. The activity of this enzyme preparation was remarkably inhibited by metal reagents such as α, α'-bipyridyl, ethylenediaminetetraacetic acid, ο-phenanthroline and nitroso-R-salt, and the inhibition was reversed by the addition of ferrous ions, and reactivation increased by the further addition of reducing agents, but not by the reducing agent such as ascorbic acid alone.
3. Enzyme and ferrous ion combination was proved to be necessary for the activity of the gentisic acid oxidase as the results of experiments using several combinations of iron-free enzyme, substrate and ferrous ions.
4. The loss of activity of iron-free enzyme could be recovered by the addition of ferrous ion. Other metalic ions and reducing agents tested were not effective.
5. Participation of SH-radical in the enzymesubstrate combination was presumed from the inhibition pattern by SH-reagents such as p-chloromercury-benzoate.
The authors wish to express their sincere thanks to Honorary Prof. K. Sakaguchi for his continued guidance throughout this research.