No abstract

1. The influence of halogen substituents on the 1,2-dioxygenation of catechols was investigated. The results obtained with the two isoenzymes pyrocatechase I and pyrocatechase II from the haloarene-utilizing Pseudomonas sp. B 13 and the pyrocatechase from benzoate-induced cells of Alcaligenes eutrophus B.9 were compared. 2. Substituents on catechol were found to interfere with O2 binding by the two isoenzymes from Pseudomonas sp. B 13, whereas the Km value for catechol kept constant at different O2 concentrations. 3. Electron-attracting substituents decreased the Km values for catechols. 4. Results from binding studies with substituted catechols demonstrated narrow stereospecificities of pyrocatechase I from pseudomonas sp. B 13 and the pyrocatechase from alcaligenes eutrophus B.9. In contrast, a low steric hindrance by substituents in the binding of catechols with pyrocatechase II was observed. 5. Low pK'1 values of substituted catechols resulted in low Michaelis constants. 6. Electron-attracting substituents such as halogen decreased the reaction rates of catechol 1,2-dioxygenation. The correlation of the Vmax. values observed with pyrocatechase II from Pseudomonas sp. B 13 with the substituent constant sigma+ (Okamoto--Brown equation) was distinctly greater than with Hammett's sigma values. The corresponding logVmax. against sigma+ correlation for pyrocatechase I was considerably disturbed by steric influences of the substituents.

1. Two catechol 1,2-dioxygenases, pyrocatechase I and pyrocatechase II, were found in 3-chlorobenzoate-grown cells of Pseudomonas sp. B 13. The latter enzyme showed high relative activities with 3- and 4-chlorocatechol compared with catechol. 2. In benzoate-grown cells, only pyrocatechase I was induced. It was purified 29-fold with a final specific activity of 20 mumol of catechol oxygenated/min per mg of protein and an overall yield of 22%. Because of the instability of pyrocatechase II on chromatography and dialysis, no increase of specific activity was obtained during the purification experiments. 3. Molecular weights of pyrocatechase I and pyrocatechase II were 82000 and 67000 respectively. 4. For both pyrocatechases the pH optimum was found to be at 8.0.5. Inhibitions of the two pyrocatechases by Cu2+ and Hg2+ ions and p-chloromercuribenzoate were different. The effect on pyrocatechase I after incubation for 20 h with the heavy metals was decreased by addition of 1 mM-2-mercaptoethanol to the reaction mixture. The inhibition of pyrocatechase II was even enhanced under these conditions. 6. Extradiol cleavage of 3-methylcatechol in addition to intradiol fission at a ratio of 1:14 was observed only with pyrocatechase I.

Gottingen, Federal Republic of Germany 3-Chlorobenzoate-grown cells of Pseudomonas sp. B13 readily cometabolized monofluorobenzoates. A catabolic pathway for the isomeric fluorobenzoates is proposed on the basis of key metabolites isolated. Only 4-fluorobenzoate was utilized and totally degraded after a short period of adaptation. The isoenzymes for total degradation of chlorocatechols, being found during growth with 3chlorobenzoate or 4-chlorophenol, were not induced in the presence of fluorobenzoates. Correspondingly, only the ordinary enzymes of the benzoate pathway were detected in 4-fluorobenzoate-grown cells. Ring cleavage of 3-fluorocatechol was recognized as a critical step in 3-fluorobenzoate degradation. 2-Fluoro-cis,cismuconic acid was identified as a dead-end metabolite from 2-and 3-fluorobenzoate catabolism. During 2-fluorobenzoate cometabolism, fluoride is eliminated by the initial dioxygenation.