The interaction of substrates with a 4-methoxybenzoate 0-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 Pseudomoms 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 a t 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 a t 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.The biodegradation of aromatic 0-alkyl ethers in bacteria is generally initiated by monooxygenase systems requiring reduced pyridine nucleotides and oxygen. An activated form of oxygen attacks the &-carbon atom and the methyl group is oxidatively eliminated probably via an unstable semiacetalUnlike the well-known nonspecific 0-dealkylating system in mammalian liver microsomes [2,3], the bacterial monooxygenases have been found to exhibit rather high specificity for the phenolic ethers on which the bacteria were grown.Cartwright et al. [4-61 have reported the existence of 3-and 4-methoxybenzoate demethylases in cell extracts from Pseudomonas fluorescens and Pseudomonas strain P 6. These enzymes again are different by pH-optima, sensitivity to inhibitors and stability against dialysis from the 4-methoxybenzoate 0-demethylating system which we have isolated from Pseudomonas putida [ 11. Recent investigations by Ribbons [7,8] on 3-methoxybenzoic acid 0-demethylases from P. aeruginosa and P. testosteroni in agreement with the results of Cartwright and our findings indicate that these monooxygenases may be multienzyme systems with similar structures.We have highly purified and characterized an iron-containing flavoprotein and an iron-sulphur protein (EPR at g = 1.91) as components of the 0-demethylating enzyme system from P. putidu grown on 4-methoxybenzoate [9]. There is no indication for the participation of a cytochrome, although Cartwight et al. [iO,ii] have recently described cytochrome P450 and another heme component in 0-demethylase systems from Noeardia NH 1 and P. fluorescens Tp, respectively. ...

Regulation of Rubisco, Anionic Modulators, Specificity and Mechanism o f Action o f Dianionic EffectorsCatalysis and regulation o f C 0 2 fixation differ in a characteristic manner in their response to anionic modifiers and the polarity of the reaction medium. Monovalent inorganic anions inhibit catalysis and C 0 2-activation of the D-ribulose 1,5-bisphosphate carboxylase/oxygenase from spinach, whereas the activity and binding o f NADPH and effector sugar phosphates are affected only at appreciably higher concentrations. In contrast such modulators with a dianion structure stimulate C 0 2 fixation by an increase of the affinity o f the enzyme for the activator C 0 2 and stabilization of the reactive carbamate. Structure-activity studies revealed a broad specificity o f the enzyme for these regulatory effects. Essentially amino groups are involved in these processes. Certain organic solvents, as methanol or acetone, stimulate C 0 2 fixation by a similar modification of the C 0 2 activation centers, as induced by dianionic effectors. These results infer that such effects are due to a decrease of the polarity at the regulatory centers of the enzyme and a concomitant change of the pK of the active lysine responsible for the binding o f the activator C 0 2. A correlation of effector binding and activity demonstrates that already low, non-saturating concentrations o f such modifiers induce high activation levels of the carboxylase and prevent the dissociation o f the activated ternary complex. It is discussed that the central problem concerning the catalytical competence o f D-ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the presence of active site directed dianionic effectors can be solved kinetically.

Single-phase Pr 2 CBr was prepared by heating a mixture of PrBr 3 , Pr and C (1 : 5:3) to 1140 • C for 18 d. The crystal structure was investigated by X-ray single crystal diffraction (space group P6 3 /mmc, a = 3.8071 (3), c = 14.7787(12)Å). In the structure the Pr atoms form C-centered octahedra condensed into Pr 2 C sheets via common edges; these sheets are separated by the Br atoms which are in a trigonal prismatic environment of Pr atoms. Pr 2 CBr is a black shiny compound with metallic conductivity. It is a ferromagnet with T c = 13.8(5) K.

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