Murine liver homogentisate 1,2-dioxygenase. Purification to homogeneity and novel biochemical properties.
1. The murine liver enzyme homogenisate 1,2-dioxygenase (HGO) is purified 330-fold. The molecular mass, as determined by gel filtration, is approximately 149 kDa. SDS/PAGE under strongly reducing conditions reveals a single band at 49 kDa, whose concentration increases during the purification. 2. HGO has a pI = 8. The pH optimum for activity in vitro is 6.1. 3. The Km for its natural substrate HGA is 188 microM, whereas the Kd for the ferrous ion is determined at 19 microM. Fe2+ is an absolutely obligate cofactor and cannot be replaced by other divalent cations. Incubating the enzyme with Fe2+ plus one of other divalent cations Zn2+, Cu2+, Co2+ or Ni2+ in equimolar concentrations inhibits HGO, whereas addition of Ca2+ and Mn2+ has no effect. Ascorbate probably acts as a reducing agent, protecting Fe2+ from spontaneous oxidation or by reversing its oxidation. Ascorbate has a great potential to stabilize the assay system. 4. The activation energy determined by an Arrhenius plot is 24 kJ/mol. 5. HGO consists of a single type of subunit with no intermolecular disulfide bridges and requires Fe2+ as a cofactor. This emphazises that the enzyme is familiar with the class of extradiol dioxygenases.
Schmidt
S R
SR
Department of Human Genetics, University of Würzburg, Germany.
Müller
C R
CR
Kress
W
W
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D049308:Dioxygenases; D050560:Homogentisate 1,2-Dioxygenase; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D051379:Mice; D010105:Oxygenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D010105:Oxygenases; D049308:Dioxygenases; D050560:Homogentisate 1,2-Dioxygenase; D001205:Ascorbic Acid