F. Bernhardt, N. Erdin, H. Staudinger
May 1, 1973
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Journal
European journal of biochemistry
Abstract
The interaction of substrates with a 4-methoxybenzoate O-demethylating enzyme system was studied by use of crude cell-free extracts and also by the purified enzyme system. The two components of the enzyme system, an iron-containing flavoprotein and an iron-sulphur protein, were obtained in pure state from Pseudomonas putida grown on 4-methoxybenzoate as the sole carbon source. The purified enzyme system requires NADH and oxygen as cofactors and acts on various substrates. The highest affinity is found for 4-methoxybenzoate, but also N-methyl-4-aminobenzoate is demethylated and 4-alkylbenzoates are hydroxylated at the side chain. The enzyme is rather specific for para-substituted benzoic acid derivatives whereas 3-methoxybenzoate and 4-hydroxy-3-methoxybenzoate are demethylated slowly. The enzyme is also able to hydroxylate the aromatic ring. This is shown by the isolation of 3,4-dihydroxybenzoate as the hydroxylation product of 3-hydroxy- or 4-hydroxy-benzoate, respectively. Studies on substrate binding and oxygen consumption with substrate analogues showed an absolute requirement for the carboxy group at the aromatic ring. Benzoic acid derivatives without a suitable CH-bond uncouple oxygen uptake with a concomitant formation of hydrogen peroxide. Measurements of oxygen consumption indicate that the affinity towards oxygen is substrate dependent, probably due to steric alterations as a consequence of substrate binding.