Bovine Liver Homogentisicase: Apo- and Reconstituted Holoenzymes

Bovine liver homogentisicase can exist in two forms which are interpreted as being apo-and holoenzymes. It is obtained in an unenriched form (apoenzyme) from bovine liver by acetone and ammonium sulfate fractionations and chromatography on hydroxylapatite column. This form of the enzyme can be enriched to an active form (holoenzyme) by incubation with ferrous ion under anaerobic conditions. The extent of activation is completely time dependent and the rate is extremely sensitive to the pH. Reducing substances do not affect the restoration of activity of the apoenzyme under anaerobic conditions. Properties of the apo- and holoenzymes are compared with respect to absorption spectrum, effects of reducing agents and pH on activity (or on reconstitution), stability, and molecular weight. The activity of homogentisicase is strongly inhibited by various diphenolic compounds as well as chelating agents specific for ferrous ion. However, the reconstitution of homogentisicase from the apoenzyme with ferrous ion is strongly prevented only by the substrate, homogentisate or 2,5-dihydroxybenzoate, a compound structurally similar to the substrate.