Inhibition of catechol 2,3-dioxygenase from Pseudomonas putida by 3-chlorocatechol.
Partially purified preparations of catechol 2,3-dioxygenase from toluene-grown cells of Pseudomonas putida catalyzed the stoichiometric oxidation of 3-methylcatechol to 2-hydroxy-6-oxohepta-2,4-dienoate. Other substrates oxidized by the enzyme preparation were catechol, 4-methylcatechol, and 4-fluorocatechol. The apparent Michaelis constants for 3-methylcatechol and catechol were 10.6 and 22.0 muM, respectively. Substitution at the 4-position decreases the affinity and activity of the enzyme for the substrate. Catechol 2,3-dioxygenase preparations did not oxidize 3-chlorocatechol. In addition, incubation of the enzyme with 3-chlorocatechol led to inactivation of the enzyme. Kinetic analyses revealed that both 3-chlorocatechol and 4-chlorocatechol were noncompetitive or mixed-type inhibitors of the enzyme. 3-Chlorocatechol (Ki = 0.14 muM) was a more potent inhibitor than 4-chlorocatechol (Ki = 50 muM). The effect of the ion-chelating agents Tiron and o-phenanthrolene were compared with that of 3-chlorocatechol on the inactivation of the enzyme. Each inhibitor appeared to remove iron from the enzyme, since inactive enzyme preparations could be fully reactivated by treatment with ferrous iron and a reducing agent.
D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt / Q000494:pharmacology; D050557:Catechol 2,3-Dioxygenase; D002396:Catechols / Q000378:metabolism / Q000494:pharmacology*; D002722:Chlorobenzenes / Q000378:metabolism; D049308:Dioxygenases; D004229:Dithiothreitol / Q000494:pharmacology; D005296:Ferrous Compounds / Q000494:pharmacology; D007700:Kinetics; D010105:Oxygenases / Q000037:antagonists & inhibitors*; D011549:Pseudomonas / Q000201:enzymology*
D002396:Catechols; D002722:Chlorobenzenes; D005296:Ferrous Compounds; C031136:3-methylcatechol; C031137:3-chlorocatechol; C020748:ferrous sulfate; D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; D010105:Oxygenases; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase; D004229:Dithiothreitol