SPECIFICITY OF O-DEMETHYLATION IN EXTRACTS OF THE HOMOACETOGENIC HOLOPHAGA-FOETIDA AND DEMETHYLATION KINETICS MEASURED BY A COUPLED PHOTOMETRIC ASSAY

The kinetics and specificity of O-demethylation were studied in cell-f ree extracts of the strictly anaerobic, methanethiol- and dimethylsulf ide-producing homoacetogen Holophaga foetida strain TMBS4 with methane thiol and tetrahydrofolate (H-4 folate) as methyl accepters. Extracts of cells grown with 3,4,5-trimethoxybenzoate contained an enzyme syste m that demethylated various phenyl methyl ethers with at least one ol rho-positioned hydroxyl or methoxyl group (the ortho system) and also contained a decarboxylase. Extracts of cells grown with 3,5-dihydroxya nisole contained an enzyme system with a novel specificity that demeth ylated only the meta-hydroxylated compounds 3,5-dihydroxyanisole and 3 -hydroxyanisole (the meta system) and lacked a decarboxylase. H-4 fola te-dependent demethylation produced CH3H4 folate. For a photometric in vitro assay of the meta system the NADPH-consuming phloroglucinol red uctase (PR) reaction was coupled to the phloroglucinol-yielding demeth ylation of 3,5-dihydroxyanisole. The kinetics of the indicator enzyme PR were studied. The cell extract had a high and stable specific PR ac tivity. PR was inhibited by phloroglucinol (substrate inhibition) and the substrate analogue 3,5-dihydroxyanisole. Doubling the PR activity of the coupled enzyme assay by additions of a PR-enriched fraction had no effect, showing that the PR activity supplied by cell extract did not limit reaction rates. Demethylation activity of the meta system wi th either methyl acceptor increased with the square of the protein con centration. With H-4 folate, the in vivo activity could be attained. K inetic parameters for the methyl accepters were determined.