Studies on kynurenate hydroxylase: requirement for iron and flavin nucleotide

• 1.

  1. Kynurenate hydroxylase (kynurenate, NAD(P)H:O₂ oxidoreductase (hydroxylating), EC 1.14.1.3), an enzyme which catalyzes the conversion of kynurenate to kynurenate-7,8-dihydrodiol, has been purified from Pseudomonas.

• 2.

  2. The partially purified enzyme preparation is stimulated 6–8 fold by the addition of ferrous ion. It is strongly inhibited by $\text{o-}\text{phenanthroline}$ and α,α′-dipyridyl and its activity is specifically restored by the addition of ferrous ion.

• 3.

  3. Flavin nucleotides were shown to participate in the kynurenate hydroxylase reaction. After acid ammonium sulfate treatment of the enzyme, it is active only upon the addition of flavin nucleotides.

• 4.

  4. The enzyme is unstable but is fairly well protected from inactivation by some reducing agents, heated extracts, or in an atmosphere of nitrogen. The enzyme, once inactivated by oxygen, can be reactivated almost to the original level by the addition of sodium borohydride. The effects of various reducing agents and conditions required for this reactivation have been studied. required for this reactivation have been studied.