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Purification and Properties of 3-Hydroxyanthranilate Oxygenase from Beef Kidney

Nobuaki Ogasawara 1, J. E. Gander 1, and L. M. Henderson 1

From the 1 From the Department of Biochemistry, University of Minnesota, St. Paul, Minnesota 55101

1. Beef kidney 3-hydroxyanthranilate oxygenase was purified in the inactive form, free of bound ferrous ion. Its properties are very similar to those of the liver enzyme.

2. The enzyme in the early stages of purification could be activated by acidification to about pH 4 in the presence of ferrous salts. For the most purified preparations, a sulfhydryl compound, as well as acid and iron, was required for activation. In contrast to the reports on the liver enzyme, cysteine was more effective than reduced glutathione.

3. The oxygenase was immediately activated in the presence of 6.8 m urea, Fe++, and cysteine. High concentrations of urea appeared to replace the acid in the activation process. However, the activation in 6.8 m urea was followed quickly by irreversible inactivation. Lower concentrations of urea required a few hours for activation and the resulting enzyme was stable to these concentrations of urea.

4. Metal-binding agents, including 1,10-phenanthroline, inhibited the enzyme and, at low pH or over an extended period, removed the bound iron to inactivate the oxygenase. The substrate, 3-hydroxyanthranilate, prevented the removal of iron from the active enzyme by 1,10-phenanthroline and also blocked the complete reconstitution of the active enzyme from iron-free enzyme.

5. The Michaelis constants for oxygen and 3-hydroxy-anthranilate were 3.1 x 10-4 m and 4 x 10-5 m, respectively. Kinetic treatment, varying 3-hydroxyanthranilate and oxygen concentrations, suggested that the two substrates are bound independently.

6. Picolinic and quinolinic acids, but not the other pyridine compounds tested, inhibited the reaction competitively with respect to 3-hydroxyanthranilate, but uncompetitively with respect to oxygen, suggesting that the two substrates are bound independently of one another.

Submitted on August 5, 1965


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