title: [Purification and properties of two chloridazondihydrodiol dehydrogenases from chloridazon degrading bacteria].
abstract: A cell-free extract of Chloridazon-degrading soil bacteria catalyzes the conversion of the dihydrodiol derivative of chloridazon to the corresponding catechol derivative. NAD is required as hydrogen acceptor. Chromatography of the crude extract on DEAE-cellulose results in the elution of two different enzymes (enzyme A and enzyme B, respectively) with the same catalytic capacity. Both enzymes were purified to homogeneity in disc-gel electrophoresis and their properties were compared. The molecular weight was found to be 220 000 for both enzymes. Dodecyl sulphate polyacrylamide gel electrophoresis indicated subunits of molecular weight 50 000 in both cases. The synthesis of the enzymes does not seem to be under inductive control. The two dehydrogenases differ in heat-stability, pH-optimum, Km-values for the substrate and in their sensitivity to inhibitors. Enzyme A shows relatively high heat lability, a pH-optimum at pH 9.5, and a Km-value of 0.25 mM for the dihydrodiol derivative of chloridazon. The catalytic activity of enzyme A is not influenced by p-chloromercuribenzoate or by N-bromosuccinimide. In contrast enzyme B is relatively stable at high temperatures, showing a pH-optimum of 7.0, and a Km for the dihydrodiol derivative of chloridazon of 1.0 mM. Enzyme B can be completely inhibited by even small amounts of p-chloromercuribenzoate and by N-bromosuccinimide. Striking differences were found in the substrate specificities of the two dehydrogenases. Whereas enzyme A exhibits a high specificity towards dihydrodiols derived from aromates of the chloridazon or phenazon type, enzyme B is much less specific and is also able to convert the dihydrodiols of benzene, toluene or chlorobenzene into the corresponding catechols. Both enzymes are competitively inhibited by the reaction product, the catechol of chloridazon. Other catechols differed in their inhibitory effect on the two dehydrogenases. These differences are correlated with the different substrate specificities.
mesh_terms: D001419:Bacteria / Q000201:enzymology*; D006540:Herbicides / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D010088:Oxidoreductases / Q000378:metabolism*; D011724:Pyridazines / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D006540:Herbicides; D011724:Pyridazines; C005586:pyrazon; D010088:Oxidoreductases; C019872:chloridazondihydrodiol dehydrogenase
vernacular_title: Reinigung und Eigenschaften von zwei Chloridazondihydrodiol-Dehydrogenasen aus Chloridazon-abbauenden Bakterien.