Purification and properties of L-alanine dehydrogenase of the phototrophic bacterium Rhodobacter capsulatus E1F1.
In the phototrophic nonsulfur bacterium Rhodobacter capsulatus E1F1, L-alanine dehydrogenase aminating activity functions as an alternative route for ammonia assimilation when glutamine synthetase is inactivated. L-Alanine dehydrogenase deaminating activity participates in the supply of organic carbon to cells growing on L-alanine as the sole carbon source. L-Alanine dehydrogenase is induced in cells growing on pyruvate plus nitrate, pyruvate plus ammonia, or L-alanine under both light-anaerobic and dark-heterotrophic conditions. The enzyme has been purified to electrophoretic and immunological homogeneity by using affinity chromatography with Red-120 agarose. The native enzyme was an oligomeric protein of 246 kilodaltons (kDa) which consisted of six identical subunits of 42 kDa each, had a Stokes' radius of 5.8 nm, an s20.w of 10.1 S, a D20,w of 4.25 x 10(-11) m2 s-1, and a frictional quotient of 1.35. The aminating activity was absolutely specific for NADPH, whereas deaminating activity was strictly NAD dependent, with apparent Kms of 0.25 (NADPH), 0.15 (NAD+), 1.25 (L-alanine), 0.13 (pyruvate), and 16 (ammonium) mM. The enzyme was inhibited in vitro by pyruvate or L-alanine and had two sulfhydryl groups per subunit which were essential for both aminating and deaminating activities.
Caballero
F J
FJ
Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Ciencias, Universidad de Córdoba, Spain.
Cárdenas
J
J
Castillo
F
F
D050788:Alanine Dehydrogenase; D000594:Amino Acid Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology* / Q000378:metabolism; D002244:Carbon / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004790:Enzyme Induction / Q000187:drug effects; D005970:Glutamate Synthase / Q000378:metabolism; D007700:Kinetics; D009584:Nitrogen / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D013438:Sulfhydryl Compounds; D002244:Carbon; D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase; D005970:Glutamate Synthase; D009584:Nitrogen