Purification and characterization of N-methylalanine dehydrogenase.
Cell free extracts of Pseudomonas MS previously have been shown to carry out the synthesis of a novel amino acid, N-methylalanine (Kung, H.F., and Wagner, C. (1970) Biochim. Biophys. Acta 201, 513-516). An enzyme has been isolated from this organism which is responsible for the synthesis of N-methylalanine. The stoichiometry of the reaction catalyzed by this enzyme leads to the following formulation: Methylamine + pyruvate + NADPH + H-+ yields N-methylalanine + NADP-+ + H2O. This enzyme has been physically separated from alanine dehydrogenase, which is also present in these extracts. This new enzyme has been named N-methylalanine dehydrogenase. It has been purified to near homogeneity as judged by disc gel electrophoresis. Gel filtration chromatography showed that N-methylalanine dehydrogenase has an apparent molecular weight of 77,000, while electrophoresis in sodium dodecyl sulfate gave rise to a single band with a molecular weight of approximately 36,500. The enzyme is optimally active in the pH range between 8.2 and 8.6. The apparent K-m values for pyruvate, NADPH, and methylamine, respectively, are 1-5 times 10 minus 2 M, 3-5 times 10 minus 5 M, and 7.5 times 10 minus 2 M.
D000409:Alanine / Q000031:analogs & derivatives; D000594:Amino Acid Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009249:NADP; D010084:Oxidation-Reduction; D011549:Pseudomonas / Q000201:enzymology*; D013329:Structure-Activity Relationship
D009249:NADP; D000594:Amino Acid Oxidoreductases; C021566:N-methylalanine dehydrogenase; D000409:Alanine