Abstract
Summary Homogentisate oxygenase from Pseudomonas ftuorescens adapted to tyrosine has been isolated, purified and crystallized. The crystallized enzyme was homogeneous on ultracentrifugation, and its sedimentation constant was found to be 11.8 S. The molecular weight of the enzyme was calculated to be about 380 000. Like the mammalian liver enzyme, bacterial homogentisate oxygenase requires ferrous iron as a cofactor. For maximal activity, the enzyme requires at least 10 min preincubation with ferrous iron at pH 6.0. The optimal pH is 6.0. Both glutathione and ascorbate are also required for maximal activity at pH 6.0, but only the former is essential at pH 5.4. The Km values are 6 · 10−4 M for homogentisate and 1 · 10−4 M for Fe2+ at pH 6.0. Unlike the mammalian liver enzyme, the bacterial enzyme is fairly stable on aging or storage. p-Chloromercuribenzoate inhibits the enzyme with respect to ferrous iron, but non-competitively with respect to homogentisate. The properties of bacterial homogentisate oxygenase are discussed in comparison with those of the mammalian liver enzyme.
Published Version
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