title: Suicide Inactivation of Catechol 2,3-Dioxygenase from Pseudomonas putida mt-2 by 3-Halocatechols.
abstract: The inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-chloro- and 3-fluorocatechol and the iron-chelating agent Tiron (catechol-3,5-disulfonate) was studied. Whereas inactivation by Tiron is an oxygen-independent and mostly reversible process, inactivation by the 3-halocatechols was only observed in the presence of oxygen and was largely irreversible. The rate constants for inactivation (K(2)) were 1.62 x 10 sec for 3-chlorocatechol and 2.38 x 10 sec for 3-fluorocatechol. The inhibitor constants (K(i)) were 23 muM for 3-chlorocatechol and 17 muM for 3-fluorocatechol. The kinetic data for 3-fluorocatechol could only be obtained in the presence of 2-mercaptoethanol. Besides inactivated enzyme, some 2-hydroxyhexa-2,4-diendioic acid was formed from 3-chlorocatechol, suggesting 5-chloroformyl-2-hydroxypenta-2,4-dienoic acid as the actual suicide product of meta-cleavage. A side product of 3-fluorocatechol cleavage is a yellow compound with the spectral characteristics of a 2-hydroxy-6-oxohexa-2,4-dienoic acid indicating 1,6-cleavage. Rates of inactivation by 3-fluorocatechol were reduced in the presence of superoxide dismutase, catalase, formate, and mannitol, which implies that superoxide anion, hydrogen peroxide, and hydroxyl radical exhibit additional inactivation.
authors.affiliation: Gesellschaft für Strahlen- und Umweltforschung mbH, Munich, and Lehrstuhl für Chemische Mikrobiologie der Universität-Gesamthochschule, D-5600 Wuppertal 1, Federal Republic of Germany.
authors.forename: I
authors.initials: I
authors.lastname: Bartels
authors.forename: H J
authors.initials: HJ
authors.lastname: Knackmuss
authors.forename: W
authors.initials: W
authors.lastname: Reineke