Solubilization, partial purification and properties of N-methylglutamate dehydrogenase from Pseudomonas aminovorans.
1. Extracts of amine-grown Pseudomonas aminovorans contained a particle-bound N-methylglutamate dehydrogenase (EC 1.5.99.5). The enzyme was not present in succinate-grown cells, and activity appeared before growth began in succinate-grown cells which had been transferred to methylamine growth medium. 2. Membrane-containing preparations from methylamine-grown cells catalysed an N-methylglutamate-dependent uptake of O2 or reduction of cytochrome c, which was sensitive to inhibitors of the electron-transport chain. 3. N-Methylglutamate dehydrogenase activity with phenazine methosulphate or 2,6-dichlorophenol-indophenol as electron acceptor could be solubilized with 1% (w/v) Triton X-100. The solubilized enzyme was much less active with cytochrome c as electron acceptor and did not sediment in 1 h at 150000g. Solubilization was accompanied by a change in the pH optimum for activity. 4. The solubilized enzyme was partially purified by Sepharose 4B and hydroxyapatite chromatograpy to yield a preparation 22-fold increased in specific activity over the crude extract. 5. The partially-purified enzyme was active with sarcosine, N-methylalanine and N-methylaspartate as well as with N-methylglutamate. Evidence suggesting activity with N-methyl D-amino acids as well as with the L-forms was obtained. 6. The enzyme was inhibited by p-chloromercuribenzoate, iodoacetamide and by both ionic and non-ionic detergents. 2-Oxoglutarate and formaldehyde were also inhibitors. 7. Kinetic analysis confirmed previous workers' observations of a group transfer (Ping Pong) mechanism. 8. Spectral observations suggested that the partially purified preparation contained flavoprotein and a b-type cytochrome. 9. The role of the enzyme in the oxidation of methylamine is discussed.
D015086:2,6-Dichloroindophenol / Q000378:metabolism; D000588:Amines / Q000378:metabolism; D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D005971:Glutamates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008773:Methylphenazonium Methosulfate / Q000378:metabolism; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D012521:Sarcosine / Q000378:metabolism; D013053:Spectrophotometry; D013386:Succinates
D000588:Amines; D005971:Glutamates; D013386:Succinates; D008773:Methylphenazonium Methosulfate; D015086:2,6-Dichloroindophenol; D010089:Oxidoreductases, N-Demethylating; C021577:N-methylglutamate dehydrogenase; D012521:Sarcosine